Use of opioid antagonists

ABSTRACT

Embodiments of the invention provide methods of attenuating, e.g., inhibiting or reducing, cellular proliferation and migration, particularly endothelial cell proliferation and migration, including that associated with angiogenesis, as well as attenuating cancerous tumor growth and metastasis, using opioid antagonists, including, but not limited to, those that are peripherally restricted antagonists.

RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.15/643,674, filed Jul. 7, 2017, which is a continuation of U.S.application Ser. No. 13/972,129 filed Aug. 21, 2013, now U.S. Pat. No.9,717,725 issued Aug. 1, 2017, which is a continuation of U.S.application Ser. No. 12/723,339 filed Mar. 12, 2010, now U.S. Pat. No.8,518,962 issued Aug. 27, 2013, which is a continuation-in-part of U.S.application Ser. No. 11/379,010 filed Apr. 17, 2006, now U.S. Pat. No.8,524,731 issued Sep. 3, 2013, and U.S. application Ser. No. 12/723,339is also a continuation-in-part of International Application No.PCT/US2006/07892 filed Mar. 7, 2006, which claims benefit under 35U.S.C. 119(e) of the filing dates of U.S. Provisional Application Nos.60/659,193 filed Mar. 7, 2005, 60/725,703 filed Oct. 12, 2005,60/731,009 filed Oct. 28, 2005, and 60/760,851 filed Jan. 20, 2006, theentire disclosures of which are incorporated herein by reference.

STATEMENT REGARDING FEDERAL SPONSORED RESEARCH OR DEVELOPMENT

This invention was supported in part by National Institutes of Healthgrants: DE12322; DE00470; and DE015830. The United States government hascertain rights in this invention.

FIELD OF INVENTION

The invention relates to methods of attenuating migration and/orproliferation of endothelial cells, especially associated with tumors,as well as attenuating tumor growth and/or metastasis, utilizing opioidantagonists.

INTRODUCTION

Cellular proliferation is a normal ongoing process in all livingorganisms that involves numerous factors and signals that are delicatelybalanced to maintain regular cellular cycles. Whether or not mammaliancells will grow and divide is determined by a variety of feedbackcontrol mechanisms, which includes the availability of space in which acell can grow, and the secretion of specific stimulatory and inhibitoryfactors in the immediate environment.

Unwanted proliferation or hyperproliferation, i.e., cell division andgrowth that is not a part of normal cellular turnover and metabolism, isassociated with cells in an abnormal state or neoplasm in which thecells have lost responsiveness to normal growth controls. Pathologicalproliferation of cells is often associated with angiogenic, metastaticand invasive processes.

The process of angiogenesis results in the formation of new bloodvessels. Under normal physiological conditions, animals, includinghumans, undergo angiogenesis only in very specifically restrictedsituations. For example, angiogenesis is normally observed in woundhealing, fetal and embryonic development, and formation of the corpusluteum, endometrium and placenta.

Angiogenesis, however, can be stimulated and harnessed by some neoplasms(e.g., tumors) to increase nutrient uptake. The development ofmalignancy is associated with this tumor-induced angiogenesis.Angiogenesis is essential for the growth of solid tumors beyond 2-3 mmin diameter and for tumor metastasis (Folkman, 1995; reviewed in Boucket al., 1996). In contrast to normal angiogenesis, which leads toanastomoses and capillary maturation, angiogenesis associated withneoplasia is a continuous process. Endothelial cells are activated bynearby neoplastic cells to secrete not only vascular endothelial growthfactor (VEGF) which stimulates angiogenesis, but also matrixmetalloproteases (MMP) which degrade the surrounding extracellularmatrix. The endothelial cells then invade the extracellular matrix wherethey migrate, proliferate, and organize to form new blood vessels, whichsupport neoplasm growth and survival.

Metastasis occurs when cancer cells leave the original tumor site andmigrate to other parts of the body via the bloodstream or the lymphaticsystem. The metastatic process involves complex intracellular mechanismsthat alter cancerous cells and their interactions with surrounding cellsand tissues. To begin, malignant cells break away from the primary tumorand attach to and degrade proteins that make up the surroundingextracellular matrix (ECM), which separates the tumor from adjoiningtissue. By degrading these proteins, cancer cells are able to breach theECM and escape. Thus invasion of the local extracellular matrix by tumorcells is the first step in metastasis. Further establishment ofmetastasis relies upon successful penetration of the circulatory orlymphatic system, and vessel extravasation at a secondary organ. Thecomplexity of these processes and the lack of understanding ofunderlying molecular mechanism have hindered effective therapeuticinterventions that prevent and/or inhibit metastasis of cancers. Currentmethods of treating metastases of cancers are often inadequate.

It is noted that suppression of any one of the steps of and/or factorsinvolved in angiogenesis could inhibit the formation of new vessels, andtherefore, affect tumor growth and generation of metastases. Indeed, ithas been estimated that the elimination of a single endothelial cellcould inhibit the growth of 100 tumor cells (Thorpe et al., 1995).Antibodies raised against the angiogenic factor VEGF have been shown tosuppress tumor growth in vivo (Kim et al., 1993).

BRIEF DESCRIPTION OF THE INVENTION

There is a need for improved methods of treating cancer, especiallymetastatic cancers. It has surprisingly been found that opioidantagonists can inhibit endothelial proliferation and migrationassociated with angiogenesis, and inhibit growth and metastasis ofcancerous tumors.

Important embodiments of the present invention provide methods ofattenuating, e.g., inhibiting or reducing, cellular proliferation andmigration, particularly endothelial cell proliferation and migration,including that associated with angiogenesis, using opioid antagonists(also known as opioid receptor antagonists), including, but not limitedto, those that are peripherally restricted antagonists.

According to one aspect of the invention, a method of treatment isprovided. The method involves administering to a subject with a disordercharacterized by unwanted migration or proliferation of endothelialcells an effective amount of an opioid antagonist. The treatment mayinhibit one or both of migration and proliferation. The unwantedmigration or proliferation of endothelial cells can be unwantedmigration or proliferation of vascular endothelial cells, including, butnot limited to, unwanted neovascularization or angiogenesis. Examples ofunwanted neovascularization include, but are not limited to,neovascularization associated with cancer and ocular neovascularization.The treated disorder can be any disorder characterized by unwantedmigration or proliferation of endothelial cells. Such importantdisorders are cancer, sickle cell anemia, vascular wounds, proliferativeretinopathies, and unwanted endothelial cell proliferation in thekidneys and the lung.

In important embodiments, the opioid antagonist is a peripheral opioidantagonist. Peripheral opioid antagonists include, but are not limitedto, quaternary morphinan derivatives, piperidine-N-alkylcarboxylates,and quaternary benzomorphans, and related derivatives as well as certainPEGylated antagonists. One such important peripheral opioid antagonistis methylnaltrexone. Another opioid antagonist is alvimopan. Inimportant embodiments, the effective amount of an antagonist is suchthat the subject has effective circulating blood plasma levels of theopioid antagonist continuously for at least 1 week, at least 2 weeks, atleast 3 weeks and, preferably, at least 4 weeks.

In some embodiments, the invention also includes the co-administrationof the opioid antagonists with agents that are not opioid antagonists,but which are nonetheless useful in treating disorders characterized byunwanted migration or proliferation of endothelial cells. Examples ofsuch agents include anticancer agents, antineovascularization agents(for example, anti-VEGF monoclonal antibody), antidiabetes agents,anti-sickle cell agents, wound healing agents, and anti-endothelial cellproliferative agents.

It will be understood that the subjects treated in accordance withmethods of the invention may or may not be on concurrent opioid therapy,depending on the particular disorder the subject has, the severity ofthe disorder, and the need the subject has for pain management. In someembodiments, the subject is taking concurrent opioid therapy. In someembodiments, the subject is not taking concurrent opioid therapy. Insome embodiments, the subject is taking concurrent chronic opioidtherapy. In some embodiments, the subject is not taking concurrentchronic opioid therapy.

According to another aspect of the invention, a method of inhibitingVEGF activity in endothelial cells is provided. The method involvescontacting the cells with an effective amount of an opioid antagonist.

According to yet another aspect of the invention, a method of inhibitingexogenous opioid-induced cellular migration or proliferation inendothelial cells is provided. The method involves contacting the cellswith an effective amount of an opioid antagonist.

According to a further aspect of the invention, a method of inhibitingRhoA activation in endothelial cells is provided. The method involvescontacting the cells with an effective amount of an opioid antagonist.

According to any of the foregoing embodiments, the opioid antagonistsuitably is a peripheral opioid antagonist, more suitably, a mu-opioidantagonist (also known as a mu-opioid receptor (MOR) antagonist, or aMOR antagonist), and most suitably, methylnaltrexone.

In some embodiments, the invention provides methods of attenuatingmigration and/or proliferation of endothelial cells of a tumor orcancer, comprising contacting the cells with an antimigratory oranti-proliferative amount of an opioid antagonist. In another aspect,the invention provides methods of attenuating angiogenesis associatedwith cancer. Thus, the invention contemplates treating, for example, ahuman cancer patient by attenuating angiogenesis in a cancerous tissueof a patient, through administering to the cancer tissue of the patientan effective amount of an opioid antagonist.

In other embodiments, the invention also provides a method of treatingabnormal neovascularization, comprising administering to a patient inneed of such treatment, an amount of an opioid antagonist effective toinhibit the formation of blood vessels.

Importantly, further embodiments of the invention also include a methodof attenuating tumor progression and metastasis in animal tissues,comprising contacting tumor cells or tissues with a growth-inhibitingamount of an opioid antagonist, and a method of attenuatingproliferation of hyperproliferative cells in a subject, comprisingadministering to the subject at least one opioid antagonist, in anamount which is effective to attenuate proliferation of thehyperproliferative cells. In another embodiment, the method involvesadministering a peripheral opioid antagonist, especially a peripheralmu-opioid antagonist and, in particular, a quaternary derivative ofnoroxymorphone, to a subject with cancer, e.g., lung cancer, whether ornot the cancer involves angiogenesis, to treat or inhibit thedevelopment or recurrence of the cancer. Cancers not involvingangiogenesis include those that do not involve the formation of a solidtumor fed by neovasculature. Certain blood cell cancers fall into thiscategory, for example: leukemias (cancer of the leukocytes or whitecells), lymphomas (arising in the lymph nodes or lymphocytes), and somecancers of the bone marrow elements. The method thus involvesadministering to a subject with a disorder characterized byhyperproliferation of cells an effective amount of a peripheral opioidantagonist. In one embodiment, the cells are cancer cells. The cancercells may be cancer cells associated with angiogenesis or they may beunassociated with angiogenesis. In some embodiments, the peripheralopioid antagonist is methylnaltrexone.

In further embodiments, the invention provides methods of treatingcancer, wherein a peripheral opioid antagonist and at least one othertherapeutic agent that is not an opioid or opioid antagonist areco-administered to the patient. Suitable therapeutic agents includeanticancer agents (including chemotherapeutic agents and antineoplasticagents), as well as other antiangiogenesis agents. It has beendiscovered that opioid antagonists co-administered with variousanticancer drugs, radiotherapy or other antiangiogenic drugs can giverise to a significantly enhanced anti-proliferative effect on cancerouscells, thus providing an increased therapeutic effect, e.g., employingperipheral opioid antagonists to certain tumors can potentiate theirresponse to other therapeutic regimens. Specifically, a significantlyincreased antiproliferative effect, including but not limited to, asignificantly increased antiangiogenic effect, is obtained withco-administered combinations as described in more detail below. Not onlycan an existing regimen be enhanced, but new regimens are possible,resulting, for example, in lower concentrations of the anticancercompound, a lower dosing of radiation, or lower concentration of otherantiangiogenic drugs, compared to the treatment regimes in which thedrugs or radiation are used alone. There is the potential, therefore, toprovide therapy wherein adverse side effects associated with theanticancer or other antiangiogenic drugs or radiotherapy areconsiderably reduced than normally observed with the anticancer or otherantiangiogenic drugs or radiotherapy when used alone.

Thus, in one aspect of the invention, a method of treatment is provided,which involves administering to a subject with a disorder characterizedby hyperproliferation of cells an effective amount of an opioidantagonist and an anticancer agent, radiation, or an antiangiogenicagent. In one embodiment, the cells are cancer cells. In anotherembodiment, the opioid antagonist is a peripheral opioid antagonist,especially a peripheral mu-opioid antagonists (i.e., a peripheral MORantagonist). In another aspect, the peripheral opioid antagonist ismethylnaltrexone. In further aspect of the invention, a method ofreducing the risk of recurrence of a cancer in a subject after medicalintervention is provided. The method involves administering to thesubject before, during or after the medical intervention an effectiveamount of an opioid antagonist and an anticancer agent, radiation, or anantiangiogenic agent. In one embodiment, the opioid antagonist is aperipheral opioid antagonist. In one embodiment, the peripheral opioidantagonist is methylnaltrexone.

In another aspect of the invention, the opioid antagonists are usedperi-operatively. By “peri-operatively,” it is meant immediately before(e.g., in preparation for), during, and/or immediately after a surgeryor a surgical or endoscopic procedure, e.g., colonoscopy,gastrolaparoscopy, and especially a surgery or surgical procedureinvolving the removal of a tumor. The opioid antagonists act toattenuate the recurrence of and/or the metastasis of the tumor,especially that arising from angiogenesis associated therewith.

It is anticipated that the opioid antagonist may suitably be given in acontinuous dosing regimen, e.g., a regimen that maintains a minimum, andeven more suitably, a relatively constant blood level, such as bycontinuous infusion, including via implant. It is also contemplated thatthe opioid antagonist mat be suitably injected directly intratumorallyor released from an intratumoral implant. It is further contemplatedthat the methods of the present invention may have prophylactic value incertain disorders associated with abnormal angiogenesis. Thus, theinvention provides a method of preventing the appearance orre-appearance of a disorder in a mammal, the disorder beingcharacterized by unwanted endothelial cell migration or proliferation,including abnormal angiogenesis, comprising administering to a mammal inneed of such treatment, an effective amount of an opioid antagonist,wherein the disorder is a cancer, sickle cell anemia, ocular neovasculardiseases, diabetes, ocular retinopathy, or other unwanted endothelialproliferation in kidneys, eye or lung. It will therefore be understoodthat, as used herein, treating a subject with a disorder characterizedby unwanted endothelial cell proliferation or migration includestreating a subject with an active disorder to inhibit or cure thedisorder and treating a subject to inhibit a disorder from re-occurring.For example, the subject may have had a solid tumor removed, and thesubject may receive the treatment to inhibit the tumor from recurrence.

In attenuating cell proliferation, the invention provides a method oftreatment of abnormal cell proliferation of a cell expressing vascularendothelial growth factor (VEGF) in a mammal which comprisesadministering to the mammal a therapeutically effective amount of anopioid antagonist. The invention also includes a method of treatingcancerous tissue in a subject comprising administering to the subject anamount of an opioid antagonist sufficient to inhibit VEGF production inthe cancerous tissue, as well as a method of treating angiogenicdisease, the method comprising contacting a tissue or a population ofendothelial cells with a composition comprising an amount of at leastone of an opioid antagonist under conditions effective to inhibitVEGF-induced angiogenesis and to treat angiogenic disease.

In another aspect, the present invention provides a method of inhibitingor reducing angiogenesis, particularly opioid-induced angiogenesis,e.g., of tumor cells, by administering or providing an opioidantagonist, particularly a peripheral opioid antagonist, to cellsundergoing angiogenesis. In a further aspect, the invention providesmethods of treating opioid-induced angiogenesis in patients receivingopioid treatment or in patients where the angiogenesis is induced byendogenous opioids. The former group is typically cancer patients onopioid-based pain management. The methods comprise administering anopioid antagonist to a patient in an antiangiogenic amount, e.g., anamount sufficient to inhibit or reduce the opioid-induced angiogenesis.In those patients receiving opioid treatment, the opioid and theperipheral opioid antagonist may be co-administered. Peripheral opioidantagonists can, thus, be used to inhibit or reduce the angiogeniceffects of opioids on tumor cells, and attenuate the growth of a tumor.

Suitable opioid antagonists generally include heterocyclic aminecompounds that belong to several different classes of compounds. Forexample, one class is suitably tertiary derivatives of morphinan, and inparticular, tertiary derivatives of noroxymorphone. In one embodiment,the tertiary derivative of noroxymorphone is, e.g., naloxone ornaltrexone.

Suitable peripheral opioid antagonists are also generally heterocyclicamine compounds that may belong to several different classes ofcompounds. For example, one class is suitably quaternary derivatives ofmorphinan, and in particular, quaternary derivatives of noroxymorphone.In one embodiment, the quaternary derivative of noroxymorphone is, e.g.,N-methylnaltrexone (or simply methylnaltrexone). In other embodiments,certain substituted tertiary morphinans are also suitably peripherallyrestricted in activity. Another class of peripheral antagonists isN-substituted piperidines. In one embodiment, the N-piperidine is apiperidine-N-alkylcarbonylate, such as, e.g., alvimopan. Another classof compounds which may be of value in the methods of the presentinvention is quaternary derivatives of benzomorphans. Also, available asperipheral opioid antagonists are certain polymeric conjugates of opioidantagonists, e.g., PEGylated opioid antagonists.

In some embodiments of the invention, the opioid antagonist may be amu-opioid antagonist (i.e., a MOR antagonist). In other embodiments, theopioid antagonist may be a kappa opioid antagonist. The invention alsoencompasses administration of more than one opioid antagonist, includingcombinations of mu antagonists, combinations of kappa antagonists andcombinations of mu and kappa antagonists, for example, a combination ofmethylnaltrexone and alvimopan, or a combination of naltrexone andmethylnaltrexone.

In other embodiments, it is contemplated that increased expressionlevels of mu-opioid receptor (MOR) in subjects samples is indicative ofresponsiveness to treatment with MOR antagonist. Expression levels arecorrelated to therapeutic efficiency of MOR antagonist treatment.Increased expression levels can be a measure of cancer prognosis, i.e.,of developing cancer or a recurrence of cancer.

In further embodiments, the invention provides methods of treatingopioid-induced angiogenesis in patients receiving an opioid, wherein aperipheral opioid antagonist and at least one other therapeutic agentthat is not an opioid or opioid antagonist are co-administered to thepatient. Suitable therapeutic agents include anticancer agents(including chemotherapeutic agents and antineoplastic agents), as wellas other antiangiogenesis agents.

In yet another aspect, the invention provides a method of reducing therisk of recurrence of a cancer or tumor after medical intervention (suchintervention to include but not be limited to surgery, e.g., pulmonarysurgery, surgical and endoscopic procedures, e.g., colonoscopy,gastrolaparoscopy, chemotherapy, etc.), comprising co-administering to acancer patient an opioid antagonist. Thus, the invention contemplates,for example, a method of minimizing the post-operative recurrence of,e.g., breast cancer, in a patient, comprising administering to thepatient an effective amount of an opioid antagonist. Peripheral opioidantagonists in accordance with the present invention, e.g., MNTX, canalso inhibit VEGF, platelet-derived growth factor (PDGF), or sphingosine1-phosphate (S1P)-stimulated or induced cell proliferation in theendothelial cells.

BRIEF DESCRIPTION OF DRAWINGS

The invention may be better understood and appreciated by reference tothe detailed description of specific embodiments presented herein inconjunction with the accompanying drawings of which:

FIG. 1 is a bar graph of dose-dependent inhibition of humanmicrovascular endothelial cell (HMVEC) migration, depicting the resultsfrom Example 1.

FIG. 2 is a bar graph of dose-dependent inhibition of humanmicrovascular endothelial cell migration, depicting the results fromExample 2.

FIG. 3 is a bar graph of dose-dependent inhibition of HMVEC migrationusing MNTX and MNTX+DAMGO.

FIG. 4 is a bar graph of dose-dependent inhibition of HMVEC migrationusing naloxone and naloxone+DAMGO.

FIG. 5 is a bar graph of dose-dependent effect of M3G and M6G on HMVECmigration.

FIG. 6 is a photomicrograph that shows morphine induced endothelial cellmigration in the presence and absence of MNTX. Panel A=Control, PanelB=MS (morphine sulfate), Panel C=MNTX, and Panel D=MS+MNTX. Arrows areshown in Panel A to highlight several cells that have successfullymigrated across the membrane.

FIG. 7 is a bar graph of percent proliferation (A) and migration (B) ofhuman pulmonary microvascular endothelial cells in the presence of VEGF,morphine and DAMGO with or without MNTX.

FIG. 8 is (A) a panel of immunoblots indicating the tyrosinephosphorylation (activation) of anti-VEGF R.1 (Flt-1) and 2 (Flk-1)using immunoprecipitated VEGF R.1 or 2 and anti-phospho-tyrosine inhuman pulmonary microvascular endothelial cells in the presence of VEGF,morphine and DAMGO with or without MNTX and (B) a bar graph of percentproliferation and migration of human pulmonary microvascular endothelialcells in the presence of VEGF, morphine and DAMGO with or without VEGF Rinhibitor.

FIG. 9 is a panel of immunoblots indicating RhoA activation usinganti-RhoA in human pulmonary microvascular endothelial cells in thepresence of VEGF, morphine and DAMGO with or without MNTX (A) or VEGF Rinhibitor (B).

FIG. 10 is a panel of immunoblots (A) of anti-RhoA of human pulmonarymicrovascular endothelial cells in the presence of scramble siRNA(targeting no known human mRNA sequence) or RhoA siRNA and a bar graphof percent proliferation (B) and migration (C) of human pulmonarymicrovascular endothelial cells in the presence of VEGF, morphine andDAMGO with or without scramble siRNA (targeting no known human mRNAsequence) or RhoA siRNA.

FIG. 11 is a schematic diagram summarizing the mechanism of MNTX effectson angiogenesis.

FIG. 12 is a bar graph of percent proliferation above control ofpulmonary microvascular endothelial cells in the presence of S1P, VEGF,PDGF, morphine and DAMGO with or without MNTX.

FIG. 13 is a bar graph of percent migration above control of pulmonarymicrovascular endothelial cells in the presence of S1P, VEGF, PDGF,morphine and DAMGO with or without MNTX.

FIG. 14 is a bar graph of percent proliferation above control ofpulmonary microvascular endothelial cells in the presence of S1P, VEGF,PDGF, morphine and DAMGO with scramble (control) siRNA or with mu-opioidreceptor siRNA.

FIG. 15 is a bar graph of percent migration above control of pulmonarymicrovascular endothelial cells in the presence of S1P, VEGF, PDGF,morphine and DAMGO with scramble (control) siRNA or with mu-opioidreceptor siRNA.

FIG. 16 is a panel of immunoblots indicating phosphorylation(activation) of the mu-opioid receptor using immunoprecipitatedmu-opioid receptor and (A, C) anti-phospho-serine, (B, D)anti-phospho-threonine of human pulmonary microvascular endothelialcells in the presence of morphine, DAMGO, S1P, VEGF, PDGF with MNTX (C,D) or without MNTX (A, B); (E) is an immunoblot of anti-mu-opioidreceptor.

FIG. 17 is an anti-RhoA immunoblot of (A, B) activated RhoA and (C)total RhoA of human pulmonary microvascular endothelial cells in thepresence of morphine, DAMGO, S1P, VEGF, PDGF with MNTX (B) and withoutMNTX (A).

FIG. 18 is a panel of immunoblots of top panel: (A, B)anti-phospho-tyrosine, (C) anti-VEGF R and bottom panel: (A, B)anti-phospho-tyrosine, (C) anti-PDGF R, of human pulmonary microvascularendothelial cells in the presence of morphine, DAMGO, VEGF (top panel)or PDGF (bottom panel) with MNTX (B in each panel) or without MNTX (A ineach panel).

FIG. 19 is a panel of immunoblots indicating tyrosine phosphorylation(activation) of the S1P₃ receptor using immunoprecipitated S1P₃ receptorand (A, B) anti-phospho-tyrosine, (C) anti-S1P₃ R, of human pulmonarymicrovascular endothelial cells in the presence of morphine, DAMGO, andS1P with MNTX (B) or without MNTX (A).

FIG. 20 is a bar graph of percent proliferation above control ofpulmonary microvascular endothelial cells in the presence of S1P, VEGF,PDGF, morphine and DAMGO with scramble (control) siRNA or with RhoAsiRNA.

FIG. 21 is a bar graph of percent migration above control of pulmonarymicrovascular endothelial cells in the presence of S1P, VEGF, PDGF,morphine and DAMGO with scramble (control) siRNA or with RhoA siRNA.

FIG. 22 is an schematic diagram summarizing the mechanism of MNTXeffects on RhoA activation and angiogenesis.

FIG. 23 is a graph of percent proliferation above control ofmicrovascular endothelial cells in the presence of VEGF with MNTX, with5-FU and with a combination of MNTX and 5-FU.

FIG. 24 is a graph of percent migration above control of microvascularendothelial cells in the presence of VEGF with MNTX, with Bevacizumaband with a combination of MNTX and Bevacizumab.

FIG. 25 is a bar graph of the effects of MNTX, 5-FU, and a combinationof both on SW 480 human colorectal cancer cell line.

FIG. 26 is a bar graph of the effects of MNTX, 5-FU, and a combinationof both on HCTII6 human colorectal cancer cell line.

FIG. 27 is a bar graph of the effects of MNTX, 5-FU, and a combinationof both on MCF-7 human breast cancer cell line.

FIG. 28 is a bar graph of the effects of MNTX, 5-FU, and a combinationof both on non-small lung cancer cell (NSLCC) line.

FIG. 29 A-C, wherein (A) is a bar graph of the inhibition of Lewis LungCarcinoma (LLC) cell proliferation using siRNA-induced knock-down ofmu-opioid receptor (MOR) expression; (B) is a bar graph of theinhibition of serum-induced invasion by LLC cells using siRNA-inducedknock-down of MOR siRNA or control RNA; and (C) is an in vivo imagingwith LLC cells transfected with either MOR siRNA or control RNA.

FIGS. 30 A and B are a set of bar graphs depicting (A) dose-dependentinhibition of LLC cell proliferation; and (B) invasion usingmethylnaltrexone (MNTX).

FIG. 31 A-D, wherein (A) is a photograph of primary LLC tumor growth inwild-type and MOR knock-out (MOR1-/-) mice; (B) is a bar graph depictingthe quantification of LLC primary tumor volume in wild-type (WT) and MORknock-out (MOR-/-) mice; (C) is an in vivo image showing metastaticlesions of wild-type (WT) and MOR knock-out (MOR1-/-) mice implantedwith LLC cells; and (D) is a bar graph showing the quantification ofmetastatic lesions in wild-type (WT) and MOR knock-out (MOR-/-) micevisualized using ProSense 680 (Pro) and MMPSense (MMP) probes.

FIG. 32 A-C are a series of graphs showing (A) the inhibition of LLCtumor growth; (B) formation of lung micrometastases; and (C)macrometastases in mice implanted with LLC cells and treated with MNTX.

FIG. 33 is a photomicrograph of H&E stained lung sections showing adecrease in lung macrometastases (arrows) in mice implanted with LLCcells in the flank and treated with MNTX.

FIG. 34 is a photograph of gross lung pathology showing the decrease inlung macrometastases (arrows) in mice implanted with LLC cells in theflank and treated with MNTX.

DETAILED DESCRIPTION OF THE INVENTION

Embodiments of the present invention provide methods of attenuatingabnormal or undesirable migration and/or proliferation of endothelialcells. As such, some embodiments provide methods for attenuatingangiogenesis in a tissue or an organ of a subject by the use of opioidantagonists, and a novel approach for treating angiogenic relateddiseases and other hyperproliferative diseases in mammals. For example,as described above, solid tumors rely on the generation of new bloodvessels for nutrients to reach the cells within the tumor. The growthfactors required for angiogenesis can be produced by the tumor cells oralternatively, exogenous factors, such as opioids, which can stimulatenew blood vessel growth. Embodiments of the present invention by the useof opioid antagonists provide a novel therapeutic approach to thetreatment of such tumors, wherein the generation of new blood vesselswithin the tumor, rather than the tumor cells themselves, is the target.Such treatment is not likely to lead to the development of resistanttumor cells.

Described herein are opioid antagonists that inhibit proliferation andmigration induced by opioids, endogenous or exogenous, and growthfactors, such as VEGF, PDGF, S1P etc. Mu-opioid antagonists andperipheral opioid antagonists, in particular, showed a substantialefficacy in inhibiting opioid- and growth factor-induced proliferationand migration of endothelial cells. The peripheral opioid antagonistmethylnaltrexone (MNTX) inhibited both opioid- and growth factor-inducedproliferation and migration in a concentration dependent manner. Inaddition, naloxone also inhibited opioid-induced endothelial migration.It should be noted, however, that the naloxone inhibition ofDAMGO-induced migration of endothelial cells occurred at a relativelyhigh micromolar concentration of naloxone. Furthermore, it has now beendiscovered that opioid antagonists, and the peripheral opioid antagonistMNTX in particular, inhibit agonist-induced endothelial cell (EC)proliferation and migration via inhibition of receptor phosphorylationand/or transactivation and subsequent inhibition of RhoA activation. Theagonists can be opioids, exogenous and/or endogenous, angiogenic factors(VEGF), and other proliferation and/or migration stimulating factors(PDGF, S1P, S1P₃ receptor, RhoA, etc). These results suggest thatinhibition of angiogenesis by opioid antagonists can be a usefultherapeutic intervention for, among other disorders, cancer.

The present invention also provides methods of attenuating abnormal orundesirable proliferation of cancer cells per se. This aspect of theinvention is useful in situations involving the presence or absence ofangiogenic activity. The absence of angiogenic activity is evidenced byone or more of the following characteristics: nonsolid tumors or tumorswhere there is repulsion of existing blood vessels and/or absence ofmicrovessels within the tumor, limited growth, for example, up to about1 mm in diameter in vivo, at which time further expansion is stopped,harmless to the host until it switches to an angiogenic phenotype, etc.Nonangiogenic tumors can be completely avascular or they can containempty lumen without red blood cells. The gross difference between thenonangiogenic and angiogenic tumors (i.e. white vs. red tumors) is mostlikely due to the reactive hyperemia that accompanies the onset of bloodflow after the angiogenic switch is completed in a previously hypoxictumor. Examples of nonangiogenic tumor lineages include, but are notlimited to, breast adenocarcinoma, osteosarcoma, glioblastoma, embryonickidney tumors etc. There are many factors that could play a role intumor dormancy and the rate-determining step for tumor expansion ofnonangiogenic tumors could be governed by lack of angiogenesis and/ordifferentiation programs, tumor cell survival, immune response to thehost etc. Although some nonagiogenic tumors never switch to anangiogenic phenotype, many undergo spontaneous transformation into anangiogenic and harmful phenotype. Therefore, treatment of nonangiogenictumors is of therapeutic significance.

Cancers not involving angiogenesis include those that do not involve theformation of a solid tumor fed by neovasculature. Certain blood cellcancers can fall into this category, for example: leukemias, includingacute lymphocytic leukemia (ALL), acute myelogenous leukemia (AML),chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML),and hairy cell leukemia; lymphomas (arising in the lymph nodes orlymphocytes) including Hodgkin lymphoma, Burkitt's lymphoma, cutaneouslymphoma, cutaneous T-cell lymphoma, follicular lymphoma, lymphoblasticlymphoma, MALT lymphoma, mantle cell lymphoma, Waldenstrom'smacroglobulinemia, primary central nervous system lymphoma; and somecancers of the bone marrow elements including Ewing's sarcoma andosteosarcoma.

Before any embodiments of the invention are explained in detail, it isto be understood that the invention is not limited in its application tothe details of the structure and function of the invention set forth inthe following description or illustrated in the appended figures of thedrawing. The invention is capable of other embodiments and of beingpracticed or carried out in various ways. Also, it is to be understoodthat the phraseology and terminology used herein is for the purpose ofdescription and should not be regarded as limiting. The use of termssuch as “including,” “comprising,” or “having” and variations thereofherein is meant to encompass the item listed thereafter and equivalentsthereof as well as additional items.

Unless otherwise noted, technical terms are used according toconventional usage. As used herein, however, the following definitionsmay be useful in aiding the skilled practitioner in understanding theinvention:

“Subject” refers to mammals such as humans, dogs, cats, and horses.

“Chronic opioid use” refers to and is characterized by the need forsubstantially higher levels of opioid to produce the therapeutic benefitas a result of prior opioid use, as is well known in the art. Chronicopioid use as used herein includes daily opioid treatment for a week ormore or intermittent opioid use for at least two weeks.

“Alkyl” refers to an aliphatic hydrocarbon group which is saturated andwhich may be straight, branched or cyclic having from 1 to about 10carbon atoms in the chain, and all combinations and subcombinations ofchains therein. Exemplary alkyl groups include methyl, ethyl, n-propyl,isopropyl, butyl, isobutyl, see-butyl, tert-butyl, pentyl, hexyl,heptyl, octyl, nonyl and decyl.

“Lower alkyl” refers to an alkyl group having 1 to about 6 carbon atoms.“Alkenyl” refers to an aliphatic hydrocarbon group containing at leastone carbon-carbon double bond and having from 2 to about 10 carbon atomsin the chain, and all combinations and sub combinations of chainstherein. Exemplary alkenyl groups include vinyl, propenyl, butenyl,pentenyl, hexenyl, heptenyl, octenyl, nonenyl and decenyl groups.

“Alkynyl” refers to an aliphatic hydrocarbon group containing at leastone carbon-carbon triple bond and having from 2 to about 10 carbon atomsin the chain, and combinations and sub combinations of chains therein.Exemplary alkynyl groups include ethynyl, propenyl, butenyl, pentenyl,hexenyl, heptenyl, octenyl, nonenyl and decenyl groups.

“Alkylene” refers to a bivalent aliphatic hydrocarbon group having from1 to about 6 carbon atoms, and all combinations and subcombinations ofchains therein. The alkylene group may be straight, branched or cyclic.There may be optionally inserted along the alkylene group one or moreoxygen, sulfur or optionally substituted nitrogen atoms, wherein thenitrogen substituent is alkyl as described previously.

“Alkenylene” refers to an alkylene group containing at least onecarbon-carbon double bond. Exemplary alkenylene groups includeethenylene (—CH═CH—) and propenylene (CH═CHCH2—).

“Cycloalkyl” refers to any stable monocyclic or bicyclic ring havingfrom about 3 to about 10 carbons, and all combinations andsubcombinations of rings therein. The cycloalkyl group may be optionallysubstituted with one or more cycloalkyl-group substituents. Exemplarycycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl,cyclohexyl, cycloheptyl and cyclooctyl groups.

“Cycloalkyl-substituted alkyl” refers to a linear alkyl group,preferably a lower alkyl group, substituted at a terminal carbon with acycloalkyl group, preferably a C₃-C₈ cycloalkyl group. Exemplarycycloalkyl-substituted alkyl groups include cyclohexylmethyl,cyclohexylethyl, cyclopentylethyl, cyclopentylpropyl, cyclopropylmethyland the like.

“Cycloalkenyl” refers to an oletinically unsaturated cycloaliphaticgroup having from about 4 to about 10 carbons, and all combinations andsubcombinations of rings therein.

“Alkoxy” refers to an alkyl-a-group where alkyl is as previouslydescribed. Exemplary alkoxy groups include, for example, methoxy,ethoxy, propoxy, butoxy and heptoxy.

“Alkoxy-alkyl” refers to an alkyl-O-alkyl group where alkyl is aspreviously described.

“Acyl” means an alkyl-CO group wherein alkyl is as previously described.Exemplary acyl groups include acetyl, propanoyl, 2-methylpropanoyl,butanoyl and palmitoyl.

“Aryl” refers to an aromatic carbocyclic radical containing from about 6to about 10 carbons, and all combinations and subcombinations of ringstherein. The aryl group may be optionally substituted with one or two ormore aryl group substituents. Exemplary aryl groups include phenyl andnaphthyl.

“Aryl-substituted alkyl” refers to a linear alkyl group, preferably alower alkyl group, substituted at a terminal carbon with an optionallysubstituted aryl group, preferably an optionally substituted phenylring. Exemplary aryl-substituted alkyl groups include, for example,phenylmethyl, phenyl ethyl and 3-(4-methylphenyl)propyl.

“Heterocyclic” refers to a monocyclic or multicyclic ring systemcarbocyclic radical containing from about 4 to about 10 members, and allcombinations and subcombinations of rings therein, wherein one or moreof the members of the ring is an element other than carbon, for example,nitrogen, oxygen or sulfur. The heterocyclic group may be aromatic ornonaromatic. Exemplary heterocyclic groups include, for example, pyrroleand piperidine groups.

“Halo” refers to fluoro, chloro, bromo or iodo.

“Peripheral,” in reference to opioid antagonists, designates opioidantagonists that act primarily on opioid receptors of physiologicalsystems and components external to the central nervous system, e.g.,they do not readily cross the blood-brain barrier in an amount effectiveto inhibit the central effects of opioids. In other words, peripheralopioid antagonists do not effectively inhibit the analgesic effects ofopioids when administered peripherally, e.g., they do not reduce theanalgesic effect of the opioids. For example, the peripheral opioidantagonist compounds employed in the methods of the present inventionexhibit high levels of activity with respect to gastrointestinal tissue,while exhibiting reduced or substantially no central nervous system(CNS) activity. The peripheral opioid antagonist compounds employed inthe present methods suitably exhibit less than about 5-15% of theirpharmacological activity in the CNS, with about 0% (e.g., no CNSactivity) being most suitable. The non-central acting characteristic ofa peripheral opioid antagonist is often related to charge, polarityand/or size of the molecule. For example, peripherally-acting quaternaryamine opioid antagonists are positively charged while the central-actingtertiary amine opioid antagonists are neutral molecules. The peripheralopioid antagonists useful in the present invention are typically muand/or kappa opioid antagonists.

As used herein, “antiangiogenesis” or “antiangiogenic” is meant to referto the capability of a molecule/compound to attenuate, e.g., inhibit,reduce or modulate, proliferation of new blood vessels, in general, and,for example, to reduce or inhibit migration and proliferation of humanmicrovascular endothelial cells in culture in the presence of certaingrowth factors. As described above, the formation of new blood vesselsby endothelial cells involves migration, proliferation anddifferentiation of the cells.

“Metastasis” is meant to refer to the ability of cells of a cancer todisseminate and form new foci of growth at noncontinguous sites (i.e.,to form metastases). See, e.g., definition of metastasis in the BasicScience of Oncology, Tammak et al., eds, McGraw-Hill (New York) 1992.

In the following description of the methods of the invention, processsteps are carried out at room temperature and atmospheric pressureunless otherwise specified. It also is specifically understood that anynumerical range recited herein includes all values from the lower valueto the upper value, e.g., all possible combinations of numerical valuesbetween the lowest value and the highest value enumerated are to beconsidered to be expressly stated in this application. For example, if aconcentration range or beneficial effect range is stated as 1% to 50%,it is intended that values such as 2% to 40%, 10% to 30%, or 1% to 3%,etc., are expressly enumerated in this specification. These are onlyexamples of what is specifically intended.

In one aspect, embodiments of the present invention relate to methods ofattenuating abnormal or undesirable cell, particularly endothelial cell,migration and/or proliferation, and angiogenesis in tissue or an organof a subject. The methods comprise providing or administering one ormore opioid antagonists in an effective amount to endothelial cells ofthe tissue or organ of a patient to inhibit endothelial cell migrationand proliferation, and angiogenesis. The angiogenesis may, in part, bethe result of receiving opioid treatment, particularly for painmanagement in cancer patients, or having high levels of endogenousopioids.

It has been observed that morphine and the mu-agonist enkephalin DAMGO([D-Ala², N-McPhe⁴, Gly⁵-ol]-enkephalin), each cause a dose-dependentincrease in migration of endothelial cells similar to that of vascularendothelial growth factor (VEGF) as measured by, e.g., a chemotaxisassay (as detailed in the examples below) or other similar assays usedto identify factors in tumor angiogenesis and the drugs that affect it.At clinically relevant concentrations of morphine, the magnitude of theeffect is approximately 70% of that which is achieved by VEGF. Thismorphine-based endothelial cell migration is attenuated by the mu-opioidantagonist methylnaltrexone (MNTX) in a dose-dependent fashion. Forexample, endothelial cell migration induced by morphine, inconcentrations as low as 10⁻⁷ M, is significantly blocked by 10⁻⁷ M MNTX(see, FIG. 2). This attenuation strongly suggesting that endothelialcell migration is mediated by morphine action on the mu-opioid receptor(MOR). As described in the examples below, the effect via the MOR ratherthan other opioid receptors is confirmed by experiments that show thehighly selective synthetic enkephalin mu-agonist DAMGO also inducesmigration. The migratory effect induced by DAMGO is also blocked by MNTX(see, FIG. 3).

In one comprehensive review (Neumann et al. Pain 1982; 13:247-52),analgesia in cancer patients was associated with a range of steady stateconcentrations of morphine and plasma ranging from 6 to 364 ng/mL. Itwas observed that an effect of morphine which causes endothelial cellmigration at 100 ng/mL is well within the clinical dose range. It,therefore, is believed by the inventors herein that a dose of MNTX whichwill maintain plasma levels of MNTX at minimum levels of between about25 and 150 ng/mL would be suitable. Such doses are attainable and arewell tolerated (Yuan et al., J Clin Pharmacol 2005; 45:538-46).

Alvimopan, another selective peripheral opioid antagonist given orally,is in late stage development for prophylaxis of postoperative ileus andthe management of opioid induced constipation (Moss et al., Pain reliefwithout side effects: peripheral opioid antagonists. In Schwartz, A. J.,editor, 33rd ASA Refresher Course in Anesthesiology, Philadelphia:Lippincott Williams & Wilkins (in press).) There is some transpassage ofalvimopan across the membrane (J. Foss, et al., Clin. Pharm. & Ther.2005, PII-90, p. 74) and it may, therefore, possess the ability toreverse some of the systemic effects of opioids without affectinganalgesia even when given orally.

Other opioid antagonists in development such as certain substitutedmorphinans, e.g., ALKS-37, and polymeric conjugated opioid antagonist,e.g., PEGylated antagonists, may be of value in the methods embodied inthe present invention.

Without being bound by any particular theory, it may be that themechanism of mu-opioid effect on endothelial cell migration occurs atthe membrane level as MNTX, unlike naloxone, is a charged molecule atphysiological pH. Morphine acts via G-protein coupled receptors, whileVEGF acts by receptor tyrosine kinases. While the actions of mu agonistsand VEGF may be independent, there is growing evidence of receptortransactivation as a mechanism. A prior investigation demonstrated thatpertussis toxin dependent GPCRs transactivate VEGF receptor-2/F1 Kl(Zeng, H. et al., J. Biol. Chem. 2003; 278:20738-45). By this manner,morphine could transactivate F11 c-l and promote an environment whereendothelial cell proliferation and tumor growth could occur. A recentstudy of MOR knockout mice infected with T241 fibrosarcoma cellsdemonstrated significant differences in the incidences of tumor growthand a 10-fold increase in F11 c-l expression in morphine-treated miceversus controls, versus no increase in morphine-treated KO mice (K.Gupta, personal communication). This provides further evidence thatmorphine stimulates endothelial cell proliferation and promotes tumorgrowth probably by trans activating FLKI phosphorylation. As such,embodiments of the present invention provide potential clinicalstrategies using MNTX as well as other peripheral opioid antagonist inconjunction with current therapies targeting VEGF. Although a directeffect by receptor transactivation is possible, a potential additionalfactor involved in the proliferation of tumors may well be the role ofchemokines as integrators of pain and inflammation. A recent review onthis subject (White et al., Nature Rev. Drug Discovery 2005; 4:834-44)also suggests a possible role for leukocytes in activating opioidreceptors.

Furthermore, it was observed that morphine, DAMGO and VEGF stimulateRhoA activation which is inhibited by opioid antagonists, such as MNTX.RhoA is an important signaling molecule involved in angiogenesis(Aepfelbacher et al., 1997; Cascone et al., 2003; Hoang et al., 2004;Liu and Sanger, 2004.) VEGF receptor transactivation is important foropiate-induced RhoA activation. Silencing RhoA expression blockedopioid- and VEGF-induced endothelial cell (EC) proliferation andmigration, demonstrating a role for RhoA activation in agonist-inducedEC angiogenic activity. The MNTX mediated attenuation of RhoA activationmay be important for the inhibitory role of MNTX on opioid and VEGFinduced angiogenesis.

Because morphine and other opioids at clinical doses enhance endothelialcell migration, embodiments of the present invention may be oftherapeutic value in opioid antagonist treatment for patients onsignificant and sustained doses of opioids that have tumors relying onthe angiogenic process. Further, while the inventors' clinicalobservations have focused on morphine, which is exogenouslyadministered, endogenous opioids, which are released by stress or pain,may also play a role in endothelial cell migration. Based on endothelialcell migration experiments detailed below in the examples, MNTX andother opioid antagonists generally are of therapeutic value as anantiangiogenic therapy even absent exogenous opioid administration (asdetailed herein). It is envisioned that the methods embodied in thepresent invention will inhibit or reduce the growth of blood vesselswithin and to a tumor. Inhibiting the growth of blood vessels withintumors prevents nutrients and oxygen from being supplied to the tumor tosupport growth beyond a certain size. Minimizing the number of bloodvessels or other tumors also lessens the probability that the tumor willmetastasize.

Embodiments of the present invention may be of therapeutic value inopioid antagonist treatment for patients who have tumors relying on theangiogenic process. Tumors that rely on angiogenic processes are solidtumors, leukemias and myelomas. Solid tumors include, but are notlimited to, adrenal cortical carcinoma, tumors of the bladder: squamouscell carcinoma, urothelial carcinomas; tumors of the bone: adamantinoma,aneurysmal bone cysts, chondroblastoma, chondroma, chondromyxoidfibroma, chondrosarcoma, fibrous dysplasia of the bone, giant celltumour, osteochondroma, osteosarcoma; breast tumors: secretory ductalcarcinoma, chordoma; colon tumors: colorectal adenocarcinoma; eyetumors: posterior uveal melanoma, fibrogenesis imperfecta ossium, headand neck squamous cell carcinoma; kidney tumors: chromophobe renal cellcarcinoma, clear cell renal cell carcinoma, nephroblastoma (Wilmstumor), kidney: papillary renal cell carcinoma, primary renalASPSCRl-TFE3 tumor, renal cell carcinoma; liver tumors: hepatoblastoma,hepatocellular carcinoma; lung tumors: non-small cell carcinoma, smallcell cancer; malignant melanoma of soft parts; nervous system tumors:medulloblastoma, meningioma, neuroblastoma, astrocytic tumors,ependymomas, peripheral nerve sheath tumors, phaeochromocytoma; ovariantumors: epithelial tumors, germ cell tumors, sex cord-stromal tumors,pericytoma; pituitary adenomas; rhabdoid tumor; skin tumors: cutaneousbenign fibrous histiocytomas; smooth muscle tumors: intravenousleiomyomatosis; soft tissue tumors: liposarcoma, myxoid liposarcoma, lowgrade fibromyxoid sarcoma, leiomyosarcoma, alveolar soft part sarcoma,angiomatoid fibrous histiocytoma (AFH), clear cell sarcoma, desmoplasticsmall round cell tumor, elastofibroma, Ewing's tumors, extraskeletalmyxoid chondrosarcoma, inflammatory myofibroblastic tumor, lipoblastoma,lipoma/benign lipomatous tumors, liposarcoma/malignant lipomatoustumors, malignant myoepithelioma, rhabdomyosarcoma, synovial sarcoma,squamous cell cancer; tumors of the testis: germ cell tumors,spermatocytic seminoma; thyroid tumors: anaplastic (undifferentiated)carcinoma, oncocytic tumors, papillary carcinoma; uterus tumors:carcinoma of the cervix, endometrial carcinoma, leiomyoma etc.

In an important embodiment of the invention, the tumors to be treatedare lung cancers, particularly non-small cell lung carcinoma's, e.g.,Lewis lung carcinoma. The compounds administered are suitably mu-opioidantagonists, especially peripheral mu-opioid antagonists, such amethylnaltrexone. Invasiveness assays as well as other known methods canassess anti-metastatic effects. As seen in the example below, cells ofLewis lung carcinoma were transplanted into the flanks of mice. The micewere administered methylnaltrexone via continuous infusion, and theresults evidence that tumor growth was attenuated as was the appearanceof micro-and micrometastases in the lungs. The reduction of tumor volumewas statistically significant (P=0.009).

The opioid antagonists in accordance with the present invention includeboth centrally and peripherally acting opioid antagonists. It iscontemplated that those antagonists of particular value are suitably theperipheral opioid antagonists. Especially suitable may be a mu-opioidantagonist, especially a peripheral mu-opioid antagonist. Opioidantagonists form a class of compounds that can vary in structure whilemaintaining the peripheral restrictive property. These compounds includetertiary and quaternary morphinans, in particular noroxymorphonederivatives, N-substituted piperidines, and in particular,piperidine-N-alkylcarboxylates, and tertiary and quaternarybenzomorphans. Peripherally restricted antagonists, while varied instructure, are typically charged, polar and/or of high molecular weight,each of which impedes their crossing the blood-brain barrier.

Examples of opioid antagonists, which cross the blood-brain barrier andare centrally (and peripherally) active, include, e.g., naloxone,naltrexone (each of which is commercially available from BaxterPharmaceutical Products, Inc.), nalmefene (available, e.g., from DuPontPharma) nalbuine, nalorphine, cyclazocine, levallorphan, cyclorphan,oxilorphan, pentazocine and naloxonazine. These may be of value inattenuating angiogenesis in the central nervous system or in patientsnot being treated for pain management or other opioid treatment.

A peripheral opioid antagonist useful for the present invention may be acompound which is a quaternary morphinan derivative, and in particular,a quaternary noroxymorphone of formula (I):

wherein R is alkyl, alkenyl, alkynyl, aryl, cycloalkyl-substituted alkylor aryl-substituted alkyl, and X⁻ is the anion, especially a chloride,bromide, iodide or methylsulfate anion. The noroxymorphone derivativesof formula (1) can be prepared, for example, according to the procedurein U.S. Pat. No. 4,176,186, which is incorporated herein by reference;see also, U.S. Pat. Nos. 4,719,215; 4,861,781; 5,102,887; 5,972,954 and6,274,591, U.S. Patent Application Nos. 2002/0028825 and 2003/0022909;and PCT publication Nos. WO 99/22737 and WO 98/25613, all of which arehereby incorporated by reference.

A compound of formula (1) of particular value is N-methylnaltrexone (orsimply methylnaltrexone), wherein R is cyclopropylmethyl as representedin formula (II):

wherein X⁻ is as described above. Methylnaltrexone is a quaternaryderivative of the opioid antagonist naltrexone. Methylnaltrexone existsas a salt, and “methylnaltrexone” or “MNTX”, as used herein, thereforeembraces salts. “Methylnaltrexone” or “MNTX” specifically includes, butis not limited to, bromide salts, chloride salts, iodide salts,carbonate salts, and sulfate salts of methylnaltrexone. Names used forthe bromide salt of MNTX in the literature include: methylnaltrexonebromide; N-methylnaltrexone bromide; naltrexone methobromide; naltrexonemethyl bromide; SC-37359, MRZ-2663-BR, andN-cyclopropylmethylnoroxy-morphine-methobromide. Methylnaltrexone iscommercially available from, e.g., Mallinckrodt Pharmaceuticals, St.Louis, Mo. Methylnaltrexone is provided as a white crystalline powder,freely soluble in water, typically as the bromide salt. The compound asprovided is 99.4% pure by reverse phase HPLC, and contains less than0.011% unquaternized naltrexone by the same method. Methylnaltrexone canbe prepared as a sterile solution at a concentration of, e.g., about 5mg/mL.

Other suitable peripheral opioid antagonists may include N-substitutedpiperidines, and in particular, piperidine-N-alkylcarboxylates asrepresented by formula (III):

wherein

R¹ is hydrogen or alkyl;

R² is hydrogen, alkyl, or alkenyl;

R³ is hydrogen, alkyl, alkenyl, aryl, cycloalkyl, cycloalkenyl,cycloalkyl-substituted alkyl, cycloalkenyl-substituted alkyl oraryl-substituted alkyl;

R⁴ is hydrogen, alkyl, or alkenyl;

A is OR^(S) or NR⁶R⁷; wherein

R⁵ is hydrogen, alkyl, alkenyl, cycloalkyl, cycloalkenyl,cycloalkyl-substituted alkyl, cycloalkenyl-substituted alkyl oraryl-substituted alkyl;

R⁶ is hydrogen or alkyl;

R⁷ is hydrogen, alkyl, alkenyl, aryl, cycloalkyl, cycloalkenyl,cycloalkyl-substituted alkyl, cycloalkenyl-substituted alkyl oraryl-substituted alkyl, or alkylene-substituted B or together with thenitrogen atom to which they are attached, R⁶ and R⁷ form a heterocyclicring selected from pyrrole and piperidine; B is

wherein R⁸ is hydrogen or alkyl;

R⁹ is hydrogen, alkyl, alkenyl, aryl, cycloalkyl, cycloalkenyl,cycloalkyl-substituted alkyl, cycloalkenyl-substituted alkyl oraryl-substituted alkyl or together with the nitrogen atom to which theyare attached, R8 and R9 form a heterocyclic ring selected from pyrroleand piperidine;

W is OR¹⁰, NR¹¹R¹², or OE; wherein

R¹⁰ is hydrogen, alkyl, alkenyl, cycloalkyl, cycloalkenyl,cycloalkyl-substituted alkyl, cycloalkenyl-substituted alkenyl, oraryl-substituted alkyl;

R¹¹ is hydrogen or alkyl;

R¹² is hydrogen, alkyl, alkenyl, aryl, cycloalkyl, cycloalkenyl,cycloalkyl-substituted alkyl, cycloalkenyl-substituted alkyl,aryl-substituted alkyl or alkylene-substituted C(═O)Y or, together withthe nitrogen atom to which they are attached, R¹¹ and R¹² form aheterocyclic ring selected from pyrrole and piperidine;

E is

alkylene-substituted (C=0)D, or —R¹³0C(═O)R¹⁴; wherein

R¹³ is alkyl-substituted alkylene;

R¹⁴ is alkyl;

D is OR¹⁵ or NR¹⁶R¹⁷ wherein

R¹⁵ is hydrogen, alkyl, alkenyl, cycloalkyl, cycloalkenyl,cycloalkyl-substituted alkyl, cycloalkenyl substituted alkyl, oraryl-substituted alkyl;

R¹⁶ is hydrogen, alkyl, alkenyl, aryl, aryl-substituted alkyl,cycloalkyl, cycloalkenyl, cycloalkyl substituted alkyl orcycloalkenyl-substituted alkyl;

R¹⁷ is hydrogen or alkyl or, together with the nitrogen atom to whichthey are attached, R¹⁶ and R¹⁷ form a heterocyclic ring selected fromthe group consisting of pyrrole or piperidine;

Y is OR¹⁸ or NR¹⁹R²⁰, wherein

R¹⁸ is hydrogen, alkyl, alkenyl, cycloalkyl, cycloalkenyl,cycloalkyl-substituted alkyl, cycloalkenyl-substituted alkyl, oraryl-substituted alkyl;

R¹⁹ is hydrogen or alkyl;

R²⁰ is hydrogen, alkyl, alkenyl, aryl, cycloalkyl, cycloalkenyl,cycloalkyl-substituted alkyl, cycloalkenyl-substituted alkyl, oraryl-substituted alkyl or, together with the nitrogen atom to which theyare attached, R¹⁹ and R²⁰ form a heterocyclic ring selected from pyrroleand piperidine;

R²¹ is hydrogen or alkyl;

and n is 0 to 4.

Particular piperidine-N-alkylcarbonylates which may be of value areN-alkylamino-3,4,4 substituted piperidines, such as alvimopanrepresented below as formula (IV):

Suitable N-substituted piperidines may be prepared as disclosed in U.S.Pat. Nos. 5,270,328; 6,451,806; 6,469,030, all of which are herebyincorporated by reference. Alvimopan is available from Adolor Corp.,Exton, Pa. Such compounds have moderately high molecular weights, azwitterion form and a polarity which prevent penetration of theblood-brain barrier.

Still other suitable peripheral opioid antagonist compounds may includequaternary benzomorphan compounds. The quaternary benzomorphan compoundsemployed in the methods of the present invention exhibit high levels ofmorphine antagonism, while exhibiting reduced, and preferablysubstantially no, agonist activity.

The quaternary benzomorphan compounds which may be employed in themethods of the present invention have the following formula (V):

wherein;

R₁ is hydrogen, acyl or acetoxy; and

R² is alkyl or alkenyl;

R is alkyl, alkenyl or alkynyl

and

X− is an anion, especially a chloride, bromide, iodide or methyl sulfateanion.

Specific quaternary derivatives of benzomorphan compounds that may beemployed in the methods of the present invention include the followingcompounds of formula (V): 2′-hydroxy5,9-dimethyl-2,2-diallyl-6,7-benzomorphanium-bromide;2′-hydroxy-5,9-dimethyl-2-n-propyl-2 allyl-6,7-benzomorphanium-bromide;2′-hydroxy-5,9-dimethy 1-2-n-propyl-2-propargy 1-6,7benzomorphanium-bromide; and2′-acetoxy-5,9-dimethyl-2-n-propyl-2-ally)-6,7 benzomorphanium-bromide.

Other quaternary benzomorphan compounds that may be employed in themethods of the present invention are described, for example, in U.S.Pat. No. 3,723,440, the entire disclosure of which is incorporatedherein by reference.

Certain substituted morphinans are also envisioned as suitable for usein the methods in accordance with the invention. For example.6-amino/peptide substituted morphinans such as, e.g., represented byformula (VI):

wherein R₃ is independently selected from the group consisting of:

-   -   (v) hydrogen;    -   (vi) aryl; substituted aryl; heteroaryl; substituted heteroaryl;    -   (vii) heterocyclic or substituted heterocyclic; and    -   (viii) —C₁, —C₈ alkyl, —C₂-C₈ alkenyl, or —C₂-C₈ alkynyl each        containing 0, 1, 2, or 3 or more heteroatoms selected from O, S,        or N; substituted —C₁-C₈ alkyl, substituted —C₂-C₈ alkenyl, or        substituted —C₂-C₈ alkynyl each containing 0, 1, 2, or 3 or more        heteroatoms selected from O, S or N; —C₃-C₁₂ cycloalkyl, or        substituted —C₃-C₁₂ cycloalkyl; —C₃-C₁₂ cycloalkenyl, or        substituted —C₃-C₁₂ cycloalkenyl;

R₄ is hydrogen, OR_(a), or NR_(b)-Q¹R_(c), where Q¹ is absent orselected from (C═O) or (SO₂);

R₅ is selected from the group consisting of: hydrogen, halogen, SR_(a),S(O)R_(a), SO₂R_(a), S(O)NR_(b)R_(c), SO₂NR_(b)R_(c), NR_(b)-Q-R_(c),CN, (C═W)NR_(b)R_(c), C(O)OR_(a), CH₂OR_(a), CH₂NR_(b)R_(c), heteroaryl,and substituted heteroaryl;

Y is hydrogen, lower alkyl, or lower alkoxy;

V₁, is C═O, SO₂, C₁-C₆ alkylene, substituted alkylene, C₂-C₆ alkenylene,C₂-C₆ alkynylene; V₂ is absent, alkylene, substituted alkylene, C₂-C₆alkenylene, C₂-C₆ alkynylene; heterocyclic, heteroaryl, aryl or C═O; V₃is absent, alkylene, substituted alkylene, C₂-C₆ alkenylene, C₂-C₆alkynylene; heterocyclic, heteroaryl, aryl or C═O; V₄ is absent,alkylene, substituted alkylene, C₂-C₆ alkenylene, C₂-C₆ alkynylene;heterocyclic, heteroaryl, aryl or C═O;

n is 1, 2, 3 or 4; wherein each repeating unit can be the same ordifferent;

Z is hydrogen, NR_(b)R_(c), (C═W)NR_(b)R_(c), NR_(a)(C═W)NR_(b)R_(c),(C═W)OH, C(O)NHOH, heteroaryl, or substituted heteroaryl;

alternatively,

can be selected from the group consisting of natural or unnatural aminoacids and peptidomimetics; and

denotes a carbon-carbon single or double bond.

Other 6-substituted morphinans are disclosed in U.S. PublishedApplication 2009/0209569, incorporated herein by reference.

Also contemplated as useful peripheral opioid antagonists arepolymerically conjugate opioid antagonists such as those described inU.S. Pat. No. 7,056,500, incorporated herein reference, having agenealogical structure of formula (VII):

POLY-Y-A_(o)   (VII)

wherein POLY is a water soluble, non-peptidic polymer, X is a linkage,preferably a hydrolytically stable linkage covalently attaching thepolymer to the opioid antagonist, and A_(o) is the opioid antagonist,and has a structure, for example, of formula (VIII):

wherein:

Y is C₁-C₆ alkyl, substituted C₁-C₆ alkyl, C₃-C₆ cycloalkyl, substitutedC₁-C₆ cycloalkyl, C₂-C₆ alkenyl, substituted C₂-C₆ alkynyl, aryl,substituted aryl, heteroaryl, substituted heteroaryl, heterocycle, andsubstituted heterocycle;

Z is H or OH;

the dashed line indicates an optional double bond; and X and POLY are asdefined above.

It is understood that for all opioid antagonists illustrated as usefulin the methods in accordance with the invention, their geometricisomers, enantiomers, diastereomers, racemates, pharmaceuticallyacceptable salts, prodrugs and solvates are also contemplated asdescribed herein.

The compounds employed in the methods of the present invention may existin prodrug form. As used herein, “prodrug” is intended to include anycovalently bonded carriers which release the active parent drugaccording to formulas (1) to (VIII) or other formulas or compoundsemployed in the methods of the present invention in vivo when suchprodrug is administered to a mammalian subject. Since prodrugs are knownto enhance numerous desirable qualities of pharmaceuticals (e.g.,solubility, bioavailability, manufacturing, etc.), the compoundsemployed in the present methods may, if desired, be delivered in prodrugform. Thus, the present invention contemplates methods of deliveringprodrugs. Prodrugs of the compounds employed in the present inventionmay be prepared by modifying functional groups present in the compoundin such a way that the modifications are cleaved, either in routinemanipulation or in vivo, to the parent compound.

Accordingly, prodrugs include, for example, compounds described hereinin which a hydroxy, amino, or carboxy group is bonded to any group that,when the prodrug is administered to a mammalian subject, cleaves to forma free hydroxyl, free amino, or carboxylic acid, respectively.

Examples include, but are not limited to, acetate, formate and benzoatederivatives of alcohol and amine functional groups; and alkyl,carbocyclic, aryl, and alkylaryl esters such as methyl, ethyl, propyl,iso-propyl, butyl, isobutyl, sec-butyl, tert-butyl, cyclopropyl, phenyl,benzyl, and phenethyl esters, and the like.

As noted, the compounds employed in the methods of the present inventionmay be prepared in a number of ways well known to those skilled in theart. All preparations disclosed in association with the presentinvention are contemplated to be practiced on any scale, includingmilligram, gram, multi gram, kilogram, multikilogram or commercialpharmaceutical scale.

Compounds employed in the present methods may contain one or moreasymmetrically substituted carbon atom, and may be isolated in opticallyactive or racemic form. Thus, all chiral, diastereomeric, racemic form,epimeric form and all geometric isomeric form of a structure areintended, unless the specific stereochemistry or isomeric form isspecifically indicated. It is well known in the art how to prepare andisolate such optically active form. For example, mixtures ofstereoisomers may be separated by standard techniques including, but notlimited to, resolution of racemic form, normal, reverse-phase, andchiral chromatography, preferential salt formation, recrystallization,and the like, or by chiral synthesis either from chiral startingmaterials or by deliberate synthesis of target chiral centers.

In some embodiments of the invention, the opioid antagonist may be amu-opioid antagonist (MOR antagonist). In other embodiments, the opioidantagonist may be a kappa opioid antagonist. The invention alsoencompasses administration of more than one opioid antagonist, includingcombinations of mu antagonists, combinations of kappa antagonists andcombinations of mu and kappa antagonists, for example, a combination ofmethylnaltrexone and alvimopan.

The methods of the present invention encompass providing a therapeuticor prophylactic role in other endothelial-based diseases, e.g., in avariety of angiogenesis and/or proliferation-related neoplastic andnon-neoplastic diseases, e.g., sickle cell disease, neovascular diseaseof the eye (such as diabetic retinopathy, neovascular glaucoma,retinopathy of prematurity, age-related macular degeneration),endothelial proliferation in the kidneys or lung and psoriasis.Non-neoplastic conditions that are amenable to treatment includerheumatoid arthritis, psoriasis, atherosclerosis, diabetic and otherproliferative retinopathies including retinopathy of prematurity,retrolental fibroplasia, neovascular glaucoma, age-related maculardegeneration, thyroid hyperplasias (including Grave's disease), cornealand other tissue transplantation, chronic inflammation, lunginflammation, nephrotic syndrome, preeclampsia, ascites, pericardialeffusion (such as that associated with pericarditis), and pleuraleffusion. For example, it has been shown that morphine inducedproliferative retinopathy in sickle cell disease (Gupta et al., personalcommunication). It is anticipated that treatment with an opioidantagonist may significantly inhibit the retinopathy, particularly withopioid-induced retinopathy in sickle cell patients that are in activeopioid therapy, and receive opioids for long periods of time, includingchronic therapy for weeks, months or even years.

The methods of the present invention are also envisioned to be of valuein reducing the risk of recurrence of a malignancy or neoplasm aftertreatment with other therapeutic modalities, e.g., after surgicalintervention. For example, embodiments in accordance with the presentinvention provide methods for reducing the risk of recurrence ofpostoperative cancer. The cancers may include, for example, lung cancer,breast cancer or prostate cancer, and reduced risk may be achieved byproviding to the patient suffering from such cancer an effective amountof an opioid antagonist, particularly a peripheral opioid antagonist.For example, patients undergoing breast cancer surgery had a significantdifference (fourfold) in the incidence of recurrence at 2-4 yearsdepending on whether the patients received regional or generalanesthesia (with morphine during their initial surgery).Co-administration of the opioid antagonists, especially peripheralantagonist (described herein), in accordance with the present inventionwith surgical treatment may be of value to reduce the incidence ofrecurrence of the cancer.

It is also contemplated that the invention provides a method ofinhibiting the activity of VEGF by providing to the affected cells orsubject an effective amount of an opioid antagonist under conditionssufficient to inhibit VEGF-induced angiogenesis. In other words, thecompounds in accordance with embodiments of the present invention haveVEGF-inhibitory or antagonist activity.

As also shown in the examples below, it has further been found that aperipheral opioid antagonist, MNTX, attenuates not only VEGF-inducedendothelial cell migration, but also induction of endothelial migrationand/or proliferation by other pro-migration, pro-proliferative factorssuch as platelet derived growth factor (PDGF), orsphingosine-1-phosphate (S1P). Such attenuation ranges from about 10% to60%, and provides further evidence that the methods in accordance withthe present invention have value in inhibiting pro-migration,pro-angiogenic factors.

Embodiments of the invention also encompass methods of treatingpatients, e.g., cancer patients, who are undergoing treatment withopioid agonists. Opioid agonists include, but are not limited to,morphine, methadone, codeine, meperidine, fentidine, fentanil,sufentanil, alfentanil and the like. As described above, opioid agonistsare classified by their ability to agonize one type of receptor an orderof magnitude more effectively than another. For example, the relativeaffinity of morphine for the mu receptor is 200 times greater than forthe kappa receptor, and is therefore classified as a mu-opioid agonist.Some opioid agonists may act as agonists towards one receptor andantagonists toward another receptor and are classified asagonist/antagonists, (also known as mixed or partial agonists).“Agonist/antagonist,” “partial agonist,” and “mixed agonist” are usedinterchangeably herein. These opioids include, but are not limited to,pentazocine, butorphanol, nalorphine, nalbufine, buprenorphine,bremazocine, and bezocine. Many of the agonist/antagonist group ofopioids are agonists at the kappa receptors and antagonists at the mureceptors. Further, it is envisioned the active metabolites of opioidagonists may also be active as angiogenesis inducers. For example, themetabolites of morphine, morphine 3-glucuronide (M3G) and morphine6-glucuronide (M6G) may be active pro-angiogenic factors.

Generally, the peripheral opioid antagonists in accordance with thepresent invention may be administered in an effective amount such thatthe patient's plasma level of a peripheral opioid antagonist is in therange from 10⁻⁶ M to 10⁻⁹ M. Patient drug plasma levels may be measuredusing routine HPLC methods known to those of skill in the art.

As described in the examples below, the enkephalin analog DAMGO inducesendothelial migration. Thus, the methods in accordance with the presentinvention may be of value to patient suffering from angiogenic-relatedor hyperproliferative diseases, e.g., cancer, quite apart from treatmentwith opioid agonists.

The particular mode of administration of the opioid antagonist selectedwill depend, of course, upon the particular combination of drugsselected, the severity of the tumor progression being treated, in thecancer patient, the general health condition of the patient, and thedosage required for therapeutic efficacy. The methods of this invention,generally speaking, may be practiced using any mode of administrationthat is medically acceptable, e.g., any mode that produces effectivelevels of the active compounds without causing clinically unacceptableadverse effects. Such modes of administration include oral, rectal,topical (as by powder, ointment, drops, trans dermal patch oriontophoretic devise), transdermal, sublingual, intramuscular, infusion,intravenous, pulmonary, intramuscular, intracavity, as an aerosol, aural(e.g., via eardrops), intranasal, inhalation, intraocular orsubcutaneous. Direct injection, i.e., intratumoral injection, could alsobe used for local delivery and provide sufficiently high concentrationsof antagonist at the tumor site. Intratumoral implants may also be usedfor sustained, local delivery of antagonist at the tumor site. Oral orsubcutaneous administration may be suitable for prophylactic or longterm treatment because of the convenience of the patient as well as thedosing schedule. For ocular diseases, ophthalmic formulations may beinjected or instilled directly. Of particular value is continuousinfusion, e.g., via implant, a sustained release delivery system orcontinuous local injection.

Additionally, the opioid antagonists may be administered as anenterically coated tablet or capsule. In some embodiments, the opioidantagonist is administered by a slow infusion method or by atime-release or controlled-release method or as a lyophilized powder.

When administered, the compounds of the invention are given inpharmaceutically acceptable amounts and in pharmaceutically acceptablecompositions or preparations. Such preparations may routinely containsalts, buffering agents, preservatives, and optionally other therapeuticingredients. When used in medicine, the salts should be pharmaceuticallyacceptable, but non-pharmaceutically acceptable salts may convenientlybe used to prepare pharmaceutically acceptable salts thereof and are notexcluded from the scope of the invention. Such pharmacologically andpharmaceutically acceptable salts include, but are not limited to, thoseprepared from the following acids: hydrochloric, hydrobromic, sulfuric,nitric, phosphoric, maleic, acetic, salicylic, p-toluenesulfonic,tartaric, citric, methanesulfonic, formic, succinic,naphthalene-2-sulfonic, pamoic, 3-hydroxy-2-naphthalenecarboxylic, andbenzene sulfonic. Suitable buffering agents include, but are not limitedto, acetic acid and salts thereof (1-2% WN); citric acid and saltsthereof (1-3% WN); boric acid and salts thereof (0.5-2.5% WN); andphosphoric acid and salts thereof (0.8-2% WN).

Suitable preservatives include, but are not limited to, benzalkoniumchloride (0.003-0.03% WN); chlorobutanol (0.3-0.9% WIN); parabens(0.01-0.25% WN) and thimerosal (0.004-0.02% WN).

For ease of administration, a pharmaceutical composition of opioidantagonists may also contain one or more pharmaceutically acceptableexcipients, such as lubricants, diluents, binders, carriers, anddisintegrants. Other auxiliary agents may include, e.g., stabilizers,wetting agents, emulsifiers, salts for influencing osmotic pressure,coloring, flavoring and/or aromatic active compounds.

A pharmaceutically acceptable carrier or excipient refers to a non-toxicsolid, semi-solid or liquid filler, diluent, encapsulating material orformulation auxiliary of any type. For example, suitablepharmaceutically acceptable carriers, diluents, solvents or vehiclesinclude, but are not limited to, water, salt (buffer) solutions,alcohols, gum arabic, mineral and vegetable oils, benzyl alcohols,polyethylene glycols, gelatin, carbohydrates such as lactose, amylose orstarch, magnesium stearate, talc, silicic acid, viscous paraffin,vegetable oils, fatty acid mono glycerides and diglycerides,pentaerythritol fatty acid esters, hydroxy methylcellulose, polyvinylpyrrolidone, etc. Proper fluidity may be maintained, for example, by theuse of coating materials such as lecithin, by the maintenance of therequired particle size in the case of dispersions and by the use ofsurfactants. Prevention of the action of microorganisms may be ensuredby the inclusion of various antibacterial and antifungal agents such asparaben, chlorobutanol, phenol, sorbic acid and the like.

If a pharmaceutically acceptable solid carrier is used, the dosage formmay be tablets, capsules, powders, suppositories, or lozenges. If aliquid carrier is used, soft gelatin capsules, transdermal patches,aerosol sprays, topical cream, syrups or liquid suspensions, emulsionsor solutions may be the dosage form.

For parental application, particularly suitable are injectable, sterilesolutions, preferably nonaqueous or aqueous solutions, as well asdispersions, suspensions, emulsions, or implants, includingsuppositories. Ampoules are often convenient unit dosages. Injectabledepot form may also be suitable and may be made by formingmicroencapsule matrices of the drug in biodegradable polymers such aspolylactide-polyglycolide, poly(orthoesters) and poly(anhydrides).Depending upon the ratio of drug to polymer and the nature of theparticular polymer employed, the rate of drug release can be controlled.

Depot injectable formulations are also prepared by entrapping the drugin liposomes or microemulsions which are compatible with body tissues.The injectable formulations may be sterilized, for example, byfiltration through a bacterial-retaining filter or by incorporatingsterilizing agents in the form of sterile solid compositions which canbe dissolved or dispersed in sterile water or other sterile injectablemedia just prior to use.

For enteral application, particularly suitable are tablets, dragees,liquids, drops, suppositories, or capsules such as soft gelatincapsules. A syrup, elixir, or the like can be used wherein a sweetenedvehicle is employed.

As noted, other delivery systems may include time-release,delayed-release or sustained-release delivery system. Such systems canavoid repeated administrations of the compounds of the invention,increasing convenience to the patient and the physician and maintainsustained plasma levels of compounds. Many types of controlled-releasedelivery system are available and known to those of ordinary skill inthe art. Sustained or controlled-release compositions can be formulated,e.g., as liposomes or those wherein the active compound is protectedwith differentially degradable coatings, such as by microencapsulation,multiple coatings, etc.

For example, compounds of this invention may be combined withpharmaceutically acceptable sustained-release matrices, such asbiodegradable polymers, to form therapeutic compositions. Asustained-release matrix, as used herein, is a matrix made of materials,usually polymers, which are degradable by enzymatic or acid-basehydrolysis or by dissolution. Once inserted into the body, the matrix isacted upon by enzymes and body fluids. A sustained-release matrix may bedesirably chosen from biocompatible materials such as liposomes,polymer-based system such as polylactides (polylactic acid),polyglycolide (polymer of glycolic acid), polylactide co-glycolide(copolymers oflactic acid and glycolic acid), polyanhydrides,poly(ortho)esters, polysaccharides, polyamino acids, hyaluronic acid,collagen, chondroitin sulfate, polynucleotides, polyvinyl propylene,polyvinyl pyrrolidone, and silicone; nonpolymer system such ascarboxylic acids, fatty acids, phospholipids, amino acids, lipids suchas sterols, hydrogel release system; silastic system; peptide-basedsystem; implants and the like. Specific examples include, but are notlimited to: (a) erosional system in which the polysaccharide iscontained in a form within a matrix, found in U.S. Pat. Nos. 4,452,775,4,675,189, and 5,736,152 (herein incorporated by reference in theirentireties), and (b) diffusional system in which an active componentpermeates at a controlled rate from a polymer such as described in U.S.Pat. Nos. 3,854,480, 5,133,974 and 5,407,686 (herein incorporated byreference in their entireties). In addition, pump-based hard-wireddelivery system can be used, some of which are adapted for implantation.Suitable enteric coatings are described in PCT publication No. WO98/25613 and U.S. Pat. No. 6,274,591, both incorporated herein byreference.

Use of a long-term, sustained-release implant may be particularlysuitable for maintaining suitable drug levels. “Long-term” release, asused herein, means that the implant is constructed and arranged todeliver therapeutic levels of the active ingredient for at least 7 days,and suitably 30 to 60 days. Long-term sustained-release implants arewell-known to those of ordinary skill in the art and include some of therelease system described above.

For topical application, there are employed as nonsprayable form,viscous to semi-solid or solid form comprising a carrier compatible withtopical application and having a dynamic viscosity preferably greaterthan water. Suitable formulations include, but are not limited to,solutions, suspensions, emulsions, cream, ointments, powders, liniments,salves, aerosols, etc., which are, if desired, sterilized or mixed withauxiliary agents, e.g., preservatives, etc.

Transdermal or iontophoretic delivery of pharmaceutical compositions ofthe peripheral opioid antagonists is also possible.

Respecting MNTX specifically, aqueous formulations may include achelating agent, a buffering agent, an anti-oxidant and, optionally, anisotonicity agent, preferably pH adjusted to between 3.0 and 3.5.Suitable formulations that are stable to autoclaving and long termstorage are described in application Ser. No. 10/821,811, now publishedas 20040266806, entitled “Pharmaceutical Formulation,” the disclosure ofwhich is incorporated herein by reference.

In one embodiment, compounds of the invention are administered in adosing regimen which provides continuous dosing of the compound to asubject, e.g., a regimen that maintains minimum plasma levels of theopioid antagonist, and preferably eliminates the spikes and troughs of adrug level with conventional regimens. Suitably, a continuous dose maybe achieved by administering the compound to a subject on a daily basisusing any of the delivery methods disclosed herein. In one embodiment,the continuous dose may be achieved using continuous infusion to thesubject, or via a mechanism that facilitates the release of the compoundover time, for example, a transdermal patch, or a sustained releaseformulation. Suitably, compounds of the invention are continuouslyreleased to the subject in amounts sufficient to maintain aconcentration of the compound in the plasma of the subject effective toinhibit or reduce opioid induced angiogenesis; or in cancer patients, toattenuate growth of a tumor. Compounds in accordance with the presentinvention, whether provided alone or in combination with othertherapeutic agents (as described hereinbelow), are provided in anantiangiogenic effective amount. It will be understood, however, thatthe total daily usage of the compounds and compositions in accordancewith the present invention will be decided by the attending physicianwithin the scope of sound medical judgment. The specific therapeuticallyeffective dose level for any particular patient will depend upon avariety of factors including the disorder being treated and the severityof the disorder; activity of the specific compound employed; thespecific composition employed; the age, body weight, general health, sexand diet of the patient; the time of administration; the route ofadministration; the rate of excretion of the specific compound employed;the duration of the treatment; drugs used in combination or coincidentalwith the specific compound employed and like factors well known in themedical arts. For example, it is well within the level of ordinary skillin the art to start doses of the compound at levels lower than thoserequired to achieve the desired therapeutic effect and to graduallyincrease the dosage until the desired effect is achieved.

If desired, the effective daily dose may be divided into multiple dosesfor purposes of administration. Consequently, single dose compositionsmay contain such amounts or submultiples thereof to make up the dailydose. As noted, those of ordinary skill in the art will readily optimizeeffective doses and co-administration regimens (as described herein) asdetermined by good medical practice and the clinical condition of theindividual patient.

Generally, oral doses of the opioid antagonists, particularly peripheralantagonists, will range from about 0.01 to about 80 mg/kg body weightper day. It is expected that oral doses in the range from 1 to 20 mg/kgbody weight will yield the desired results. Generally, parenteraladministration, including intravenous and subcutaneous administration,will range from about 0.001 to 5 mg/kg body weight. It is expected thatdoses ranging from 0.05 to 0.5 mg/kg body weight will yield the desiredresults. Dosage may be adjusted appropriately to achieve desired druglevels, local or systemic, depending on the mode of administration. Forexample, it is expected that the dosage for oral administration of theopioid antagonists in an enteric ally coated formulation would be from10 to 30% of the non-coated oral dose. In the event that the response ina patient is insufficient of such doses, even higher doses (oreffectively higher dosage by a different, more localized delivery route)may be employed to the extent that the patient tolerance permits.Multiple doses per day are contemplated to achieve appropriate systemiclevels of compounds. Appropriate system levels can be determined by, forexample, measurement of the patient's plasma level of the drug usingroutine HPLC methods known to these of skill in the art.

In some embodiments of the invention, the opioid antagonists areco-administered with other therapeutic agents, including opioids. Theterm “co-administration” is meant to refer to a combination therapy byany administration route in which two or more agents are administered toa patient or subject. Co-administration of agents may also be referredto as combination therapy or combination treatment. The agents may be inthe same dosage formulations or separate formulations. For combinationtreatment with more than one active agent, where the active agents arein separate dosage formulations, the active agents can be administeredconcurrently, or they each can be administered at separately staggeredtimes. That is, agents may be administered simultaneously orsequentially (e.g., one agent may directly follow administration of theother or the agents may be give episodically, e.g., one can be given atone time followed by the other at a later time, e.g., within a week), aslong as they are given in a manner sufficient to allow both agents toachieve effective concentrations in the body. The agents may also beadministered by different routes, e.g., one agent may be administeredintravenously while a second agent is administered intramuscularly,intravenously or orally. In other words, the co-administration of theopioid antagonist compound in accordance with the present invention withanother agent is suitably considered a combined pharmaceuticalpreparation which contains an opioid antagonist and a another agent, thepreparation being adapted for the administration of the peripheralopioid antagonist on a daily or intermittent basis, and theadministration of the agent on a daily or intermittent basis. Thus, theopioid antagonists may be administered prior to, concomitant with, orafter administration of therapeutic agents. Co-administrable agents alsomay be formulated as an admixture, as, for example, in a singleformulation or single tablet. These formulations may be parenteral ororal, such as the formulations described, e.g., in U.S. Pat. Nos.6,277,384; 6,261,599; 5,958,452 and PCT publication No. WO 98/25613,each hereby incorporated by reference.

It is further contemplated that embodiments of the present methods canbe used alone or in conjunction with other treatments to control thegrowth or migration of endothelial cells in connection with the variousconditions described above. The peripheral opioid antagonist may beco-administered with another therapeutic agent that is not an opioid oranother opioid antagonist. Such suitable s therapeutic agents includeanticancer agents, e.g., chemotherapeutic agents, radiotherapy, or otherantiangiogenic agents such as suramin, or anti-VEGF mab, an endostatinor radiotherapy. It is envisioned that the opioid antagonists inaccordance with the present invention are of particular value whenco-administered with those agents that inhibit VEGF activity, e.g.,anti-VEGF mab. The anti-VEGF antibodies are useful in the treatment ofvarious neoplastic and non-neoplastic diseases and disorders, includingendometrial hyperplasia, endometriosis, abnormal vascular proliferationassociated with phakomatoses, edema (such as that associated with braintumors and Meigs' syndrome. One example of a anti-VEGF mab isbevacizumab (Avastin, Genentech) described in U.S. Pat. No. 6,884,879and W094/10202 hereby incorporated in their entirety. In a certainembodiments of the invention, MNTX is co-administered with Avastin.

In other words, the compounds of the present invention may also beuseful for the treatment of cancer in patients, as described above,either when used alone or in combination with one or more otheranticancer agents, e.g., radiotherapy and/or other chemotherapeutic,including antiangiogenic, treatments conventionally administered topatients for treating cancer. The main categories and examples of suchdrugs are listed herein and include, but are not limited to,metalloproatease inhibitors, inhibitors of endothelial cellproliferation/migration, antagonists of angiogenic growth factors,inhibitors of Integrin/Survival signaling, and chelators of copper.

In certain embodiments the compounds of the invention can be combinedwith known combinations of anticancer agents. The compounds of theinvention can be combined with an antiangiogenic agent and achemotherapeutic agent and administered to a cancer patient. Forexample, MNTX can be administered to cancer patients in combination withAvastin and 5-fluorouracil.

In one embodiment of the invention, the tumors are prostate cancer,gastrointestinal tumors such as colon or pancreatic cancer and thecompounds of the invention are co-administered with other anticanceragents as described herein.

It is anticipated that the opioid antagonists co-administered withvarious anticancer drugs, radiotherapy or other antiangiogenic drugs cangive rise to a significantly enhanced antiproliferative effect oncancerous cells, and thus providing an increased therapeutic effect,e.g., employing peripheral opioid antagonists to certain tumors canpotentiate their response to other therapeutic regimens. Specifically, asignificantly increased antiangiogenic or antihyperproliferative effectmay be obtained with the above disclosed co-administered combinations,even if utilizing lower concentrations of the anticancer agent, a lowerdosing of radiation, or other antiangiogenic drugs compared to thetreatment regimes in which the drugs or radiation are used alone.Therefore, there is the potential to provide therapy wherein adverseside effects associated with anticancer drugs, antiangiogenic drugs orradiotherapy are considerably reduced than normally observed with theanticancer drugs, antiangiogenic drugs or radiotherapy used alone inlarger doses. For example, co-administration of an opioid antagonist inaccordance with the present invention with an anti-VEGF agent, e.g.,anti-VEGF mab, may reduce the dose of the anti-VEGF agent or increasepotency or efficacy or both. Further, as detailed herein, theco-administration of an opioid antagonist in accordance with the presentinvention with other anticancer modalities may have prophylactic value.

When used in the treatment of hyperproliferative diseases, for example,compounds in accordance with the present invention may beco-administered with metalloprotease inhibitors such as for example:Marimastat, synthetic matrix metalloprotease inhibitor (MMPI), BritishBiotech; Bay 12-9566, synthetic MMPI and inhibitor tumor growth, Bayer;AG3340, synthetic MMPI, Agouron/Warner-Lambert; CGS 27023A, syntheticMMPI, Novartis; CGS 27023A, Synthetic MMPI; COL-3, synthetic MMPI,tetracycline derivative, Collagenex; AE-941 (Neovastat), naturallyoccurring MMPI, AEtema, BMS-275291, synthetic MMPI, Bristol-MyersSquibb; Penicillamine, urokinase inhibitor, NCI-NABTT.

Also, when used in the treatment of hyperproliferative diseases,compounds in accordance with the present invention may beco-administered with direct inhibitors of endothelial cellproliferation/migration such as: TNP-470 (fumagillin derivative),inhibits endothelial cell growth, TAP Pharmaceuticals; Squalamine,inhibits sodium-hydrogen exchanger, NIHE3, Magainin; Combretastatin,induction of apoptosis in proliferating endothelial cells, Oxigene;Endostatin, inhibition of endothelial cells, EntreMed; Penicillamine,blocks endothelial cell migration and proliferation, NCI-NABTT; FamesylTransferase Inhibitor (FTI), blocks endothelial cell migration andproliferation, NCI-NABTT, -L-778,123 Merck, -SCH66336 Schering-Plough,-R1l5777 Janssen.

When used in the treatment of hyperproliferative diseases, compounds inaccordance with the present invention may also be co-administered withantagonists of angiogenic growth factors such as: anti-VEGF antibody, amonoclonal antibody that inactivates VEGF, Genentech; thalidomide,blocks activity of angiogenic growth factors (bFGF, VEGF, TNF-alpha),Celgene; SU5416, blocks VEGF receptor (Flk-1/KDR) signaling (tyrosinekinase), Sugen-NCI; ribozyme (Angiozyme), attenuates mRNA of VEGFreceptors, Ribozyme Pharmaceuticals, Inc; SU6668, blocks VEGF, bFGF, andPDGF receptor signaling, Sugen; PTK787/ZK22584, blocks VEGF receptorsignaling, Novartis; Interferon-alpha, inhibition of bFGF and VEGFproduction; Suramin, blocks binding of growth factor to its receptor,NCI-NABTT.

When used in the treatment of hyperproliferative diseases, compounds ofthe present invention may also be co-administered with drugs thatinhibit endothelial-specific Integrin/Survival signaling: Vitaxin,antibody to alpha-v-beta3 integrin present on endothelial cell surface,Ixsys; EMD121974, small molecule blocker of integrin present onendothelial cell surface, Merck KGaA.

When used in the treatment of hyperproliferative diseases, compounds ofthe present invention may be co-administered with chelators of copper,such as: penicillamine, sulfhydryl group binds copper; clears copperthrough urinary excretion, NCI-NABTT; tetrathiomolybdate, thiol groupstightly bind copper, inactivate copper available to tumor, University ofMichigan Cancer Center; captopril, chelates copper and zinc; also,inhibitor of MMP and angiotensin converting enzyme, NorthwesternUniversity.

When used in the treatment of hyperproliferative diseases, compounds ofthe present invention may be co-administered with angiogenesisantagonists with distinct mechanisms: CAI, inhibitor of calcium influx,NCI; ABT-627, endothelin receptor antagonist, Abbott, NCI; CM10l/ZDO101,group B Strep toxin that selectively disrupts proliferating endotheliumby interaction with the (CM201) receptor, CarboMed/Zeneca;Interleukin-12, induction of interferon-gamma, down-regulation of IL-10,induction of IP-10, M. D. Anderson Cancer Center/Temple University,Temple University, Genetics Institute, Hoffman LaRoche; IM862, blocksproduction of VEGF and bFGF; increases production of the inhibitorIL-12, Cytran; PNU-145156E, blocks angiogenesis induced by Tat protein,Pharmacia and Upjohn.

When used in the treatment of hyperproliferative diseases, compounds ofthe present invention may be co-administered with chemotherapeuticagents such as, for example, alpha interferon, COMP (cyclophosphamide,vincristine, methotrexate and prednisone), etoposide, mBACOD(methortrexate, bleomycin, doxorubicin, cyclophosphamide, vincristineand dexamethasone), PRO-MACE/MOPP (prednisone, methotrexate (w/leucovinrescue), doxorubicin; cyclophosphamide, paclitaxol, docetaxol,etoposide/mechlorethamine, vincristine, prednisone and procarbazine),vincristine, vinblastine, angioinhibins, TNP-470, pentosan polysulfate,platelet factor 4, angiostatin, LM-609, SU-I0l, CM-lOl, Techgalan,thalidomide, SP-PG and the like.

Anticancer agents which may be co-administered with compounds inaccordance with the present invention also suitably includeantimetabolites (e.g., 5-fluoro-uracil, methotrexate, fludarabine),antimicrotubule agents (e.g., vincristine, vinblastine, taxanes such aspaclitaxel, docetaxel), an alkylating agent (e.g., cyclophosphamide,melphalan, biochoroethylnitrosurea, hydroxyurea), nitrogen mustards,(e.g., mechloethamine, melphan, chlorambucil, cyclophosphamide andIfosfamide); nitrosoureas (e.g., carmustine, lomustine, semustine andstreptozocin;), platinum agents (e.g., cisplatin, carboplatin,oxaliplatin, JM-216, CI-973), anthracyclines (e.g., doxrubicin,daunorubicin), antibiotics (e.g., mitomycin, idarubicin, adriamycin,daunomycin), topoisomerase inhibitors (e.g., etoposide, camptothecins),alkyl sulfonates including busulfan; triazines (e.g., dacarbazine);ethyenimines (e.g., thiotepa and hexamethylmelamine); folic acid analogs(e.g., methotrexate); pyrimidine analogues (e.g., 5 fluorouracil,cytosine arabinoside); purine analogs (e.g., 6-mercaptopurine,6-thioguanine); antitumor antibiotics (e.g., actinomycin D; bleomycin,mitomycin C and methramycin); hormones and hormone antagonists (e.g.,tamoxifen, cortiosteroids) and any other cytotoxic agents, (e.g.,estramustine phosphate, prednimustine).

It will be understood that agents which can be combined with thecompounds of the present invention for the inhibition, treatment orprophylaxis of angiogenesis and/or cancers are not limited to thoselisted above, but include, in principle, any agents useful for thetreatment of opioid-induced and non-opioid-induced angiogenic diseases,tumor growth and metastasis inhibition.

It has also been understood that malignant cells often differ fromnormal cells by a differential expression of one or more proteins.Overexpression of the mu-opioid receptor (MOR) has been observed in lungcancers (Madar I, Bencherif B, Lever J, et al: Imaging delta- andmu-opioid receptors in PET in lung carcinoma patients. J. Nucl. Med. 48,207-213 (2007)). In the examples below, the inventors found anapproximately five-fold increase in MOR expression was found in Lewislung carcinoma cells.

It is contemplated that individuals with a specific MOR expression orstage of disease may respond differently to a given treatment thanindividuals lacking MOR expression. MOR can be an index to thetherapeutic efficacy of opioid antagonists in accordance with theinvention, i.e., the expression level and activity of this receptorprotein can vary depending on the effect of the opioid antagonistthereto. Such information thus can provide for the personalization ofdiagnosis and treatment.

Because MORs are differentially expressed in cancer cells in comparisonto normal cells, embodiments of the invention provide methods fordiagnosing and monitoring cancers, using the expression level of MORs.That is, the expression level of MOR, may be used for predicting ordetermining the pharmacological efficacy of an opioid antagonist.Accordingly, the invention provides methods for assessing theresponsiveness of a cancer to an opioid antagonist by detecting thepresence, or levels of, a MOR in a biological sample such as tissues,cells and biological fluids isolated from a subject.

Specifically, embodiments of methods of the invention for diagnosing ordetecting a cancer in a subject include determining the level of MOR ina test sample from said subject, and comparing the differential level ofthe MOR in the sample relative to the level in a control sample from ahealthy subject, or the level established for a healthy subject, isindicative of the disease.

Embodiments of the invention may also be suitable as methods ofmonitoring the disease progression or the treatment progress.

The expression level of MORs (or nucleic acids encoding MOR) in a testsample can be compared to a normal, standard or control level in controlsamples, wherein the levels of control samples are obtained either fromcontrol cell lines which may be identified as high, medium or low MORexpression levels, or normal tissue or body fluids from a healthysubject.

Expression levels in normal and diseased tissues, cells or samples maybe measured by various methods, for example, the expression level may bedetermined by measuring the amount by mass of the protein produced,according to a western blotting method or an ELISA method or by the useof a protein chip, or by photometric means, and the MOR as it isexpressed in a subject sample is compared with the standard values. MORexpression can be correlated with tumor reduction in the presence ofopioid antagonists, i.e., expression levels may be used to evaluatetreatment efficacy.

Such methods of the invention may also be suitable for other cancers,such as, colon, prostate, ovarian, breast, bladder renal,hepatocellular, pharyngeal, and gastric cancers.

The present invention is further explained by the following examples,which should not be construed by way of limiting the scope of thepresent invention.

EXAMPLES Example 1: Endothelial Cell Migration Assay

The antiangiogenic activity of the peripheral opioid antagonists inaccordance with the present invention was evaluated in experimentstesting the ability of the antagonist to inhibit or modulation capillaryendothelial cell migration using a modified Boyden chamber.

The endothelial cell migration assay was performed as described byLingen, M. W., Methods in Molecular Medicine, 78: 337-347 (2003), thedisclosure of which is incorporated by reference. Briefly, HumanMicrovascular Endothelial Cells (HMVEC) (Cell Systems, Kirkland, Wash.)were starved overnight in Endothelial Growth Medium (EGM) containing0.1% bovine serum albumin (BSA). Cells were then trypsinized andresuspended in Dulbecco's Modified Eagle Medium (DME) with 0.1% BSA at aconcentration of 1×10⁶ cells/mL. Cells were added to the bottom of a48-well modified Boyden chamber (NeuroPore Corporation, Pleasanton,Calif.). The chamber was assembled and inverted, and cells were allowedto attach for 2 hours at 37° C. to polycarbonate chemotaxis membranes (5μm pore size) (NeuroProbe) that had been soaked in 0.1% gelatinovernight and dried. The chamber was then reinverted and the compound tobe tested at varying concentrations in quadruple, vascular endothelialgrowth factor (VEGF) (as a positive control) or vehicle were added tothe wells of the upper chamber (to a total volume of 50 mL); theapparatus was then incubated for 4 hours at 37° C. Membranes wererecovered, fixed and stained (DiffQuick, Fisher Scientific, Pittsburgh,Pa.) and the number of cells that had migrated to the upper chamber per10 high power fields were counted. Background migration to DME+0.1% BSAwas subtracted and the data reported as the number of cells migrated per10 high power fields (400 times). Each substance was tested inquadruplicate in each experiment, and all experiments were repeated toleast twice. VEGF (R&D System, Minneapolis, Minn.) was used as apositive control at a concentration of 200 pg/mL. The optimalconcentration for VEGF was determined previously by dose-responseexperiments (data not shown). The compounds tested as described abovewere morphine, naloxone, methylnaltrexone, and the combination ofmethylnaltrexone and morphine. The concentrations of each testedsubstance ranged for 0.001 to 10.0 μM. The concentration of the morphinewas constant at 0.1 μM. The results are shown in FIG. 1.

FIG. 1 shows that morphine increased migration in aconcentration-dependent manner The co-addition of methylnaltrexone andmorphine, however, decreased migration in a concentration-dependentmanner. Neither methylnaltrexone or naloxone alone affected migration.

Example 2: Endothelial Cell Migration Assay

Another set of experiments was conducted in accordance with theprocedure described in Example 1. In this set of experiments,methylnaltrexone and the combination of methylnaltrexone and morphinewas again tested for ability to inhibit endothelial cell migration. Themethylnaltrexone concentrations when tested alone varied from 0.001 to10.0 μM. In combination, the concentrations of methylnaltrexone variedfrom 0.001 to 10.0 μM, while the morphine concentration remainedconstant at 0.1 μM as described in Example 1. The results are shown inFIG. 2.

FIG. 2 shows the combination of methylnaltrexone and morphine decreasedmigration in a concentration-dependent manner, while methylnaltrexonealone did not affect migration.

Example 3: Endothelial Cell Migration Induced by DAMGO

The drugs used in this study were [D-Ala 2, N-McPhe4, Gly5-ol]enkephalin or DAMGO (Sigma, St. Louis, Mo.); naloxone (Sigma, St. Louis,Mo.); N-methylnaltrexone bromide or methylnaltrexone (MallinckrodtSpecialty Chemicals, Phillipsburg, N.J.). The endothelial cell migrationassay was performed as previously described (9). Human dermalmicrovascular endothelial cells (Cell Systems, Kirkland, Wash.) werestarved overnight in media containing 0.1% bovine serum albumin (BSA),harvested, resuspended into Dulbecco's Modified Eagle's media (DME) with0.1% BSA, and plated on a semi-porous gelatinized membrane in a modifiedBoyden chamber (Nucleopore Corporation, Pleasanton, Calif.). Testsubstances were then added to the wells of the upper chamber and cellswere allowed to migrate for four hours at 37° C.

Membranes were recovered, fixed, and stained and the number of cellsthat had migrated to the upper chamber per ten high power fields countedby a blinded observer. Background migration to DME+0.1% BSA wassubtracted and the data reported as the number of cells migrated per 10high power fields (400×). Each substance was tested in quadruplicate ineach experiment and all experiments were repeated at least twice. Theconcentration of DAMGO was 1 μM, VEGF (R&D Systems, Minneapolis, Minn.)was used as a positive control at a concentration of 200 pg/mL. Theoptimal concentration for VEGF was determined previously bydose-response experiments (data not shown).

The results are shown in FIG. 3 which shows that methylnaltrexone andDAMGO decreased migration in a concentration-dependent manner. FIG. 4illustrates similar results with naloxone and DAMGO. The inactivemorphine metabolite M3G exerts no angiogenic activity while M6G known toact at the mu receptor exhibited a concentration dependent effect onangiogenesis (FIG. 5).

Example 4: Treatment of Human and Mammalian Subjects WithMethylnaltrexone

In a first set of experiments, mice are induced to develop tumors bytransformation, inbreeding or transplantation of tumor cells. Thirty-sixmice, each bearing tumors having a volume of at least 60 mm3, arerandomly divided into three groups. The first group receives a controlsubstance comprising neither an opioid nor an opioid antagonist. Thesecond group receives an opioid, e.g., morphine administered orally at adose of 0.5 mg/kg/day. The third group receives an opioid, e.g.,morphine administered orally at a dose of 0.5 mg/kg/day, and theperipheral opioid antagonist methylnaltrexone, administered orally at adose of 5 mg/kg/day.

The compounds are administered daily for a period of eight weeks.Differences in the rate of tumor growth, tumor size, a reduction inangiogenesis in the tumor and mortality of the mice between each of thegroups are recorded. The results demonstrate a reduction in tumor growthand angiogenesis compared to controls or morphine alone.

In a second set of experiments, human cancer patients are enrolled in astudy. Enrollees in the study are controlled for age, stage of disease,treatment types and genetic and familial factors. Participants aredivided into two groups according to whether they are receiving opioids,e.g., morphine. The group receiving opioids is further randomly dividedinto two subgroups. One of the two subgroups receiving opioids receivesa peripheral opioid antagonist, e.g., methylnaltrexone administeredorally at a dose of 5 mg/kg/day for a period of eight weeks. The otherof the two subgroups receives placebo for the same period. Differencesin the rate of tumor growth, tumor size, a reduction in angiogenesis inthe tumor and mortality of the participants in each of the groups arerecorded.

Example 5: Treatment of Human and Mammalian Subjects With Alvimopan

Mice that have been induced to develop tumors are subjected to theprotocol as described in Example 3, except that the peripheral opioidantagonist is alvimopan. The results demonstrate a reduction in tumorgrowth and angiogenesis compared to controls or opioid alone.

Human cancer patients are enrolled in a study conducted as described inExample 4, except that the peripheral opioid antagonist is alvimopan.

Example 6: Therapies Comprising Co-Administration of the PeripheralOpioid Antagonist Methylnaltrexone and Second Therapeutic Agent

In a first set of experiments, mice are induced to develop tumors bytransformation, inbreeding or transplantation of tumor cells.Forty-eight mice, each bearing tumors having a volume of at least 60mm3, are randomly divided into six groups. The first group receives acontrol substance which does not comprise an opioid, an opioidantagonist, or an anticancer agent. The second group receives an opioid,e.g., morphine administered orally at a dose of 0.5 mg/kg/day. The thirdgroup receives an opioid, e.g., morphine administered orally at a doseof 0.5 mg/kg/day, and the peripheral opioid antagonist methylnaltrexone,administered orally at a dose of 5 mg/kg/day. The fourth group receivesan opioid, e.g., morphine administered orally at a dose of 0.5mg/kg/day, and the peripheral opioid antagonist methylnaltrexoneadministered orally at a dose of 5 mg/kg/day with an anticancertherapeutic agent, e.g., bevacizumab (Avastin) at a dose of 5 mg/kgevery 14 days. The sixth group receives an opioid, e.g., morphine, at adose of 0.5 mg/kg/day and an anticancer therapeutic agent, e.g.,bevacizumab (Avastin) at a dose of 5 mg/kg every 14 days.

The compounds are administered daily for a period of eight weeks.Differences in the rate of tumor growth, tumor size, a reduction inangiogenesis in the tumor and mortality of the mice in each of thegroups are recorded. The results demonstrate an enhanced result (e.g.,reduction in angiogenesis and tumor growth) for the groups administeredthe combination of opioid, opioid antagonist, and anticancer agentcompared to the other groups.

In a second set of experiments, human cancer patients receiving anopioid, e.g., morphine, an anticancer therapeutic agent, e.g.,bevacizumab (Avastin) or both are enrolled in a study. Enrollees in thestudy are controlled for age, stage and type of disease, treatment typesand genetic and familial factors. Participants receiving an opioid arerandomly divided into first and second groups; participants receiving ananticancer therapeutic agent, e.g., bevacizumab (Avastin) are randomlydivided into third and fourth groups; participants receiving an opioidplus an anticancer agent, e.g., bevacizumab (Avastin) are randomlydivided into fifth and sixth groups. The first, third and fifth groupseach receive a peripheral opioid antagonist, e.g., methylnaltrexoneadministered orally at a dose of 5 mg/kg/day for a period of eightweeks. The second, fourth and sixth groups receive placebo for the sameperiod. Differences in the rate of tumor growth, tumor size, a reductionin angiogenesis in the tumor and mortality of the participants in eachof the groups are recorded. The results demonstrate an enhanced result(e.g., reduction in angiogenesis and tumor growth) for the groupsadministered the combination of opioid, opioid antagonist, andanticancer agent compared to the other groups.

Example 7: Therapies Comprising Co-Administration of the PeripheralOpioid Antagonist Alvimopan and Second Therapeutic Agent

Mice that have been induced to develop tumors are subjected to theprotocol as described in Example 5, except that the peripheral opioidantagonist is alvimopan. The results demonstrate an enhanced result(e.g., reduction in angiogenesis and tumor growth) for the groupsadministered the combination of opioid, opioid antagonist, andanticancer agent compared to the other groups.

Human cancer patients are enrolled in a study conducted as described inExample 6, except that the peripheral opioid antagonist is alvimopan.The results demonstrate an enhanced result (e.g., reduction inangiogenesis and tumor growth) for the groups administered thecombination of opioid, opioid antagonist, and anticancer agent comparedto the other groups.

Example 8: Effect of Opioid Antagonists on Endothelial CellMigration/Proliferation

Cell culture and reagents—Human dermal microvascular endothelial cells(Cell Systems, Kirkland, Wash.) and human pulmonary microvascularendothelial cells (Clonetics, Walkersville, Md.) were cultured aspreviously described in EBM-2 complete medium (Clonetics) at 37° C. in ahumidified atmosphere of 5% CO2, 95% air, with passages 6-10 used forexperimentation (Garcia et al. 2001). Unless otherwise specified,reagents were obtained from Sigma (St. Louis, Mo.). Reagents forSDS-PAGE electrophoresis were purchased from Bio-Rad (Richmond, Calif.),Immobilon-P transfer membrane from Millipore (Millipore Corp., Bedford,Mass.). The drugs used in this study were [D-Ala2, N-MePhe\ Gly5-ol]enkephalin or DAMGO (Sigma, St. Louis, Mo.); naloxone,morphine-3-glucuronide (M3G) and morphine-6-glucuronide (M6G) (Sigma,St. Louis, Mo.); N-methylnaltrexone bromide or methylnaltrexone(Mallinckrodt Specialty Chemicals, Phillipsburg, N.J.), morphine(Baxter, Deerfield, Ill.). VEGF Receptor Tyrosine Kinase Inhibitor waspurchased from Calbiochem (San Diego, Calif.). Mouse anti-RhoA antibody,mouse anti-phosphotyrosine antibody and rho binding domain(RBD)-conjugated beads were purchased from Upstate Biotechnology (LakePlacid, N.Y.). Rabbit anti-VEGF receptor 1 (Flt-1) and anti-VEGFreceptor 2 (Flk-1) antibodies were purchased from Santa CruzBiotechnology (Santa Cruz, Calif.). Mouse anti-β-actin antibody waspurchased from Sigma (St. Louis, Mo.). Secondary horseradish peroxidase(HRP)-labeled antibodies were purchased from Amersham Biosciences(Piscataway, N.J.).

Immunoprecipitation and immunoblotting Cellular materials were incubatedwith IP buffer (50 mM HEPES (pH 7.5), 150 mM NaCl, 20 mM 30 MgCl2, 1%Triton X-100, 0.1% SDS, 0.4 mM Na3V04, 40 mM NaF, 50/lM okadaic acid,0.2 mM phenylmethylsulfonyl fluoride, 1:250 dilution of Calbiochemprotease inhibitor mixture 3). The samples were then immunoprecipitatedwith anti-VEGF receptor 1 or anti-VEGF receptor 2 IgG followed bySDS-PAGE in 4-15% polyacrylamide gels, transfer onto Immobilon™membranes, and developed with specific primary and secondary antibodies.Visualization of immunoreactive bands was achieved using enhancedchemiluminescence (Amersham Biosciences).

Determination of tyrosine phosphorylation of VEGF Receptors 1 and2—Solubilized proteins in IP buffer (see above) were immunoprecipitatedwith either rabbit anti-VEGF receptor 1 or rabbit anti-VEGF receptor 2antibody followed by SDS-PAGE in 4-15% polyacrylamide gels and transferonto Immobilon™ membranes (Millipore Corp., Bedford, Mass.). Afterblocking nonspecific sites with 5% bovine serum albumin, the blots wereincubated with either rabbit anti-VEGF receptor 1 antibody, rabbitanti-VEGF receptor 2 antibody or mouse anti-phosphotyrosine antibodyfollowed by incubation with horseradish peroxidase (HRP)-labeled goatanti-rabbit or goat anti-mouse IgG. Visualization of immunoreactivebands was achieved using enhanced chemiluminescence (AmershamBiosciences).

Construction and transfection of siRNA against RhoA—The siRNA sequencetargeting human against RhoA was generated using mRNA sequences fromGenbank™ (gi:33876092). For each mRNA (or scramble), two targets wereidentified. Specifically, RhoA target sequence 1(5′-AAGAAACTGGTGATTGTTGGT-3′) (SEQ ID NO:1), RhoA target sequence 2(5′-AAAGACATGCTTGCTCATAGT-3′) (SEQ ID NO:2), scrambled sequence 1(5′-AAGAGAAA TCGAAACCGAAAA-3′) (SEQ ID NO:3), and scramble sequence 2(5′-AAGAACCCAATTAAGCGCAAG-3′) (SEQ ID NO:4), were utilized. Sense andantisense oligonucleotides were purchased from Integrated DNATechnologies (Coralville, Iowa). For construction of the siRNA, atranscription-based kit from Ambion was used (Silencer™ siRNAconstruction kit). Human lung microvascular EC were then transfectedwith siRNA using siPORTamine™ as the transfection reagent (Ambion, Tex.)according to the protocol provided by Ambion. Cells (˜40% confluent)were serum-starved for 1 hour followed by incubated with 3 μM (1.5 μM ofeach siRNA) of target siRNA (or scramble siRNA or no siRNA) for 6 hoursin serum-free media. The serum-containing media was then added (1% serumfinal concentration) for 42 h before biochemical experiments and/orfunctional assays were conducted.

RhoA activation assay—After agonist and/or inhibitor treatment, EC aresolubilized in solubilization buffer and incubated with rho bondingdomain (RBD)-conjugated beads for 30 minutes at 4° C. The supernatant isremoved and the RBD-beads with the GTP-bound form of Rho A bound arewashed extensively. The RBD beads are boiled in SDS-PAGE sample bufferand the bound RhoA material is run on SDS-P AGE, transferred toImmobilon™ and immunqblotted with anti-RhoA antibody (Garcia et al2001).

Human dermal microvascular EC migration assay—The endothelial cellmigration assay was performed as previously described (Lingen 2002).Human dermal microvascular endothelial cells (Cell Systems, Kirkland,Wash.) were starved overnight in media containing 0.1% bovine serumalbumin (BSA), harvested, resuspended into Dulbecco's Modified Eagle'smedia (DME) with 0.1% BSA, and plated on a semi-porous gelatinizedmembrane in a modified Boyden chamber (Nucleopore Corporation,Pleasanton, Calif.). Test substances were then added to the wells of theupper chamber, and cells were allowed to migrate for 4 hr at 37° C.Membranes were recovered, fixed, and stained and the number of cellsthat had migrated to the upper chamber per 10 high-power fields wascounted by a blinded observer. Background migration to DME+0.1% BSA wassubtracted, and the data were reported as the number of cells migratedper 10 high-power fields (400×). Each substance was tested inquadruplicate in each experiment and all experiments were repeated atleast twice. Vascular endothelial growth factor (VEGF, R&D Systems,Minneapolis, Minn.) was used as a positive control at a concentration of200 pg/mL. The optimal concentration for VEGF was determined previouslyby dose-response experiments (data not shown).

Human pulmonary microvascular EC migration assay—Twenty-four Transwell™units with 8 M pore size were used for monitoring in vitro cellmigration. HPMVEC (˜1×10⁴ cells/well) were plated with varioustreatments (100 nM MNTX, 10 μM VEGF Receptor Tyrosine Kinase Inhibitoror siRNA) to the upper chamber and various agonists were added to thelower chamber (100 nM MS, DAMGO or VEGF). Cells were allowed to migratefor 18 hours. Cells from the upper and lower chamber were quantitatedusing the CellTiter96™ MTS assay (Promega, San Luis Obispo, Calif.) andread at 492 nm % migration was defined as the # of cells in the lowerchamber % the number of cells in both the upper and lower chamber. Eachassay was set up in triplicate, repeated at least five times andanalyzed statistically by Student's t test (with statisticalsignificance set at P<0.05).

Human pulmonary microvascular EC proliferation assay—For measuring cellgrowth, HPMVEC [5×10³ cells/well pretreated with various agents (100 nMMNTX, 10, μM VEGF Receptor Tyrosine Kinase Inhibitor or siRNA) wereincubated with 0.2 mL of serum-free media containing various agonists(100 nM MS, DAMGO or VEGF) for 24 h at 37° C. in 5% C02/95% air in96-well culture plates. The in vitro cell proliferation assay wasanalyzed by measuring increases in cell number using the CellTiter96™MTS assay (Promega, San Luis Obispo, Calif.) and read at 492 nm. Eachassay was set up in triplicate, repeated at least five times andanalyzed statistically by Student's t test (with statisticalsignificance set at P<0.05).

Using the endothelial cell migration assay, it was found that MS causeda concentration-dependent increase in endothelial migration. Naloxoneand MNTX alone had no effect on endothelial cell migration over a widerange of concentrations. This is demonstrated in representativephotomicrographs and quantitatively (FIGS. 6 and 1, respectively). Atclinically relevant concentrations of morphine, the magnitude of theeffect was approximately 70% of that achieved by VEGF. Endothelial cellmigration induced by morphine in concentrations as low as 10−⁷ M (FIG.2). Morphine-based endothelial cell migration was attenuated by themu-opioid antagonists naloxone and MNTX (in doses as low as 10⁻⁸ μM) ina concentration-dependent fashion, strongly suggesting that endothelialcell migration is mediated by morphine's action on the mu-opioidreceptor (MOR). That the effect is via the MOR rather than other opioidreceptors was confirmed by our observations that the highly selectivesynthetic enkephalin mu agonist DAMGO also induced migration in aconcentration dependent fashion. The effect of DAMGO was also blocked byMNTX (FIG. 3). That the inactive morphine metabolite M3G exerts noangiogenic activity, while M6G, known to act at the mu receptor,exhibits a concentration-dependent effect on angiogenesis, confirms ourhypothesis that morphine's effect on the endothelium is mediated by mureceptors (McQuay et al. 1997) (FIG. 5).

In order to assess the mechanisms of opioid and MNTX-induced effects onangiogenesis, a well-characterized EC line was used, human pulmonarymicrovascular endothelial cells (HPMVEC). In agreement with the effectson human dermal microvascular EC, it was observed that MS, DAMGO andVEGF induce HPMVEC migration which is inhibited by MNTX (FIG. 7B). Itwas shown that MS, DAMGO and VEGF also stimulate HPMVEC proliferationwhich is attenuated by MNTX (FIG. 7A).

Considering the inhibitory effects of MNTX, a mu-opioid receptorantagonist, on VEGF-induced EC proliferation and migration, the role ofopioids on VEGF receptor transactivation was examined. FIG. 8A showsthat MS and DAMGO induce tyrosine phosphorylation of both VEGF receptor1 (Flt-1) and 2 (Flk-1) which is blocked by MNTX. Further, MNTXattenuates the tyrosine phosphorylation of VEGF receptors 1 and 2induced by VEGF. These results indicate that opioids induce VEGFreceptor transactivation.

In order to address if VEGF receptor tyrosine kinase activity isrequired for opioid-induced angiogenesis, EC were pre-treated with aVEGF receptor 1 and 2 tyrosine kinase inhibitor and measuredopioid-induced EC proliferation and migration (FIG. 8B). The resultsindicate that the tyrosine kinase activity of VEGF receptors isimportant in opioid-induced EC angiogenic functions.

One important signaling molecule involved in angiogenesis is the smallG-protein, RhoA (Aepfelbacher et al. 1997; Cascone et al. 2003; Hoang etal. 2004; Liu and Senger 2004). It was observed that MS, DAMGO and VEGFstimulate RhoA activation which is inhibited by MNTX (FIG. 9A). Further,VEGF receptor transactivation is important for opioid-induced RhoAactivation (FIG. 9B). Silencing RhoA expression blocks opioid andVEGF-induced EC proliferation and migration (FIG. 10). These resultsindicate the pivotal role of RhoA activation on agonist-induced ECangiogenic activity.

Taken as a whole these findings suggest a model in which the peripheral5 mu-opioid receptor antagonist, MNTX, attenuates opioid andVEGF-induced VEGF receptor and RhoA activation. This attenuation isimportant for the inhibitory role of MNTX on opioid and VEGF-mediatedangiogenesis (FIG. 11).

Example 9: Methylnaltrexone Inhibits S1P, VEGF and PDGF-InducedAngiogenesis—Role of Receptor Transactivation

Assays were conducted according to the procedure similar to thatdescribed in Examples 1-3. It was observed that S1P, VEGF, PDGF,morphine and DAMGO induced proliferation (FIG. 12) (as measured by thecolorimetric CellTiter™ (Promega) MTS assay) and migration (FIG. 13) (asmeasured by the Transwell™ (Costar) permeable membrane filter assay (8μm pore diameter)) of EC which were inhibited by pretreatment with MNTX(0.1 μM, 1 hour). Silencing mu-opioid receptor expression (siRNA) blocksmorphine and DAMGO-induced EC proliferation (FIG. 14) and migration(FIG. 15) while also significantly inhibiting S1P, VEGF and PDGF-inducedEC proliferation (FIG. 14) and migration (FIG. 15). Immunoprecipitationfollowed by immunoblot analyses indicate that S1P, VEGF and PDGFtreatment of EC induced serine/threonine phosphorylation of themu-opioid receptor (FIG. 16) (indicating receptor transactivation) andactivation of the cytoskeletal regulatory small G-protein, RhoA (FIG.17).

Further, morphine and DAMGO treatment of EC induced tyrosinephosphorylation of the VEGF receptor (FIG. 18), PDGF receptor (FIG. 18)and S1P3 receptor (FIG. 19) along with RhoA activation. MNTXpretreatment of EC attenuated morphine, DAMGO, S1P, VEGF and PDGFinduced receptor phosphorylation events and RhoA activation. Finally,silencing RhoA expression (siRNA) blocked agonist-induced ECproliferation (FIG. 20) and migration (FIG. 21).

Taken together, these results indicate that MNTX inhibits agonistinduced EC proliferation and migration via inhibition of receptorphosphorylation/transactivation and subsequent inhibition of Rho Aactivation (FIG. 22). These results suggest that MNTX inhibition ofangiogenesis can be a useful therapeutic intervention for cancertreatment.

Example 10: Methylnaltrexone and Antiproliferative CompoundsSynergistically Inhibit VEGF-Induced Proliferation and Migration

Assays were conducted according to the procedure similar to thatdescribed in Examples 1-3. It was observed that methylnaltrexone and5-FU synergistically inhibit VEGF induced proliferation of endothelialcells.(FIG. 23). It was likewise observed that methylnaltrexone andBevacizumab synergistically inhibit VEGF induced migration ofendothelial cells.(Figure 24).

Example 11: Effects of MNTX on Various Cancer Cell Lines

The effects of MNTX on SW 480 human colorectal cancer cell line wereevaluated. As shown in FIG. 25, it was observed that MNTX itselfpossesses antiproliferation activity in SW 480 cells (**, P<0.01compared to control). In addition, MNTX enhanced 5-FU's tumoricidaleffect (*, P<0.05 compared to 5-FU 10 μM only, approx. IC50 for thiscell line). As shown in FIGS. 26, 27, and 28, respectively, similarresults were obtained in HCT116 human colorectal cancer cell line, MCF-7human breast cancer cell line, and non-small cell lung cancer cell(NSLCC) line.

Example 12: Inhibition of Mu-Opioid Receptor Signaling Decreases LungCancer Proliferation, Invasion and Metastasis

Prior studies have demonstrated that expression of the mu-opioidreceptor (MOR) is increased in patients with non-small cell lung cancer(NSCLC), a disease with poor prognosis and limited therapies. Lewis LungCarcinoma (LLC) cells were assayed for MOR expression to determinewhether this increased expression was recapitulated in this widely usednon-small cell cancer model. A ˜5-fold increased expression of MORcompared to primary lung epithelial or BEAS-2B cells was observed. Toevaluate the role of MOR in tumor proliferation and metastasis, LLCcells were treated with an MOR specific siRNA to knock-down expressionof this receptor or with the peripheral mu-opioid receptor antagonistmethylnaltrexone (MNTX) to antagonize signaling through the MOR asfollows.

Cell Culture—Lewis lung carcinoma (LLC) cells were grown in RPMI-1640media containing 10% serum. In some cases, LLC cells were treated withMOR siRNA or the peripheral MOR antagonist, methylnaltrexone (MNTX, 250nM) prior to the addition of EGF (10 ng/ml), IGF (10 ng/ml), DAMGO (1nM), morphine (1 nM) or serum (1, 5 or 10%). In vitro functional (cellproliferation and invasion) and biochemical studies (immunoblotting)were then conducted.

Animal Experiments—GFP/YFP-labeled LLC cells (1×10⁶, with or without MORsiRNA treatment) were either injected intravenously or into the flank ofwild-type (C57BL/6J) or MOR knockout mice, and tumor growth and lungmetastasis were evaluated using tumor volume measurements and/or in vivofluorescent microscopy measurements with or without intravenousinjection of ProSense and/or MMP Sense probes.

To assess the effects of MNTX, 19 C57BL/6J mice were injected with (LLC)cells (subcutaneously in the flank) (3×10⁶ cells/mouse). When theprimary tumor at the site of injection reached measurable size (˜10weeks post injection), 10 mice were then treated with continuousinfusion of methylnaltrexone (MNTX, 10 mg/kg/day) and 9 mice weretreated with PBS as a control with alzet mini pumps (100 μL totalvolume) inserted subcutaneously in the flank for 9 days. The tumor sizewas measured with calipers and the tumor volume V_(T) (mm³) wascalculated using the ellipsoid formula A²×B×π/6, where A represents thesmaller diameter.

Invasion Assays—Invasion assays were conducted using the Cell InvasionAssay Kit available from Chemicon® International (Cat. No. ECM550).Briefly, this assay was performed in an invasion chamber with a basementmembrane that was inserted into the wells of a cell culture plate. LLCcells in serum free RPMI-1640 (0.5-1.0×10⁶ cells/ml) were seeded intothe invasion chamber and placed into the cell culture well containingRPMI-1640 supplemented with fetal bovine serum (1%, 5% or 10%). Invasionwas measured as the percentage of cells that migrated through thebasement membrane. Assays were performed using LLC cells transfectedwith MOR or scramble siRNA or in the presence or absence ofmethylnaltrexone (10 nM or 100 nM).

The results demonstrated that of the experimental tumors treated,inhibition of MOR with siRNA reduced in vitro LLC proliferation (90%)(FIG. 29A) and invasion (50-75%) (FIG. 29B). Similarly, treatment of LLCcells in vitro with methylnaltrexone significantly reduced proliferation(FIG. 30A) and invasion (FIG. 30B).

To determine the role of MOR in in vivo tumor progression, metastasis inwild-type C57BL/6J mice implanted with either MOR siRNA treated LLCcells or control siRNA-treated LLC cells (3 weeks post-i.v. injection,quantitated with OV-100) was measured. In this model, in vivo lungmetastasis was reduced by ˜75% (FIG. 29C). In addition, primary LLCtumor volume was substantially reduced (>90%) in MOR knockout miceimplanted with LLC cells compared to wild-type mice (flank injection)(FIG. 31). Continuous infusion of MNTX (10 mg/kg/day) in mice withpre-existing LLC primary tumors attenuated tumor growth (FIG. 32A) aswell as the appearance of micro- (FIG. 32B) and macro-metastases in thelungs (FIG. 32C). The striking nature of the reduction in tumor growthand metastasis is exemplified by the gross pathology (FIG. 33) andHematoxylin and Eosin (H&E) stained sections (FIG. 34) of lungs takenfrom these mice. In other words, there was a statistically significantreduction (p=0.009) in tumor volume with MNTX treatment.

In conclusion, it was found that MOR knockout mice do not developsignificant tumors when injected with Lewis Lung Carcinoma as do theirwild-type controls. Further, silencing (siRNA) the expression of the MORin LLC cells inhibits lung metastasis by about 75% when injected intothe wild-type mice. Infusion of the mu-opioid antagonists, MNTX (e.g.,10 mg/kg/day) attenuates tumor growth in the wild-type mice injectedwith LLC cells by up to 90%.

Example 13: Treatment of Tumors With Varying MOR Expression Levels inHumans and Mammals

In a first set of experiments, human lung cancer cell lines in cellculture are assayed for MOR mRNA and protein expression to identifycells lines with high, medium and low MOR expression levels. These celllines are then injected into the flanks of C57BL/6 mice to establishtumors with varying MOR expression levels. When the primary tumors atthe site of injection reach measurable size (˜10 weeks post injection),tumor biopsies are taken and MOR mRNA and protein expression aremeasured to confirm that MOR expression in the tumors mirror MORexpression by the cell lines in cell culture. Mice are then treated withcontinuous infusion of a peripheral opioid antagonist, e.g.,methylnaltrexone, at dose of 10 mg/kg/day. The tumor size is measuredwith calipers and the tumor volume V_(T) (mm³) is calculated using theellipsoid formula A²×B×π/6, where A represents the smaller diameter. MORexpression levels are then correlated with tumor reduction in thepresence of opioid antagonist.

In a second set of experiments, human cancer patients are enrolled in astudy. Enrollees in the study are controlled for age, stage of disease,treatment types and genetic and familial factors. Tumor biopsies fromeach patient are assayed for MOR mRNA and protein expression andparticipants are divided into groups according MOR expression level.Each expression level group is further randomly divided into twosubgroups. One of the two subgroups receives a peripheral opioidantagonist, e.g., methylnaltrexone, administered subcutaneously at adose of 5 mg/kg/day for a period of eight weeks. The other of the twosubgroups receives placebo for the same period. Differences in the rateof tumor growth, tumor size, a reduction in angiogenesis in the tumorand mortality of the participants in each of the groups are recorded andcorrelated with MOR expression.

The correlation of MOR expression to responsiveness of the cancer totreatment with the mu-opioid antagonist may be used to set standardexpression levels against which increased MOR expression in a sample ofa patient is indicative of responsiveness to mu-opioid antagonisttreatment, of therapeutic efficiency of the antagonist and clinicalprognosis, and of the risk of developing cancer or recurrence of cancer.

In summary, embodiments in accordance with the present invention providemethods of attenuating endothelial cell migration and/or proliferationassociated with angiogenesis in a tissue or an organ, reducing tumorgrowth a volume, and/or of inhibiting metastasis of a cancer, byadministering a subject in need therefor one or more opioid antagonists,especially peripheral opioid antagonists, in an effective amount toinhibit the migration and/or proliferation and angiogenesis, tumorgrowth and/or metastasis. The methods of the present invention may alsoinvolve administering a peripheral opioid antagonist to a patientreceiving opioid treatment or not receiving such treatment. Especiallysuitable may be a peripheral mu-opioid antagonist. The present inventionalso provides methods of co-administering an opioid and a peripheralopioid antagonist to a subject in need therefore. The peripheral opioidantagonist may also be co-administered with an anticancer agent, as maythe combination of the opioid and peripheral opioid antagonist beco-administered with an anticancer agent.

While the present invention has now been described and exemplified withsome specificity, those skilled in the art will appreciate the variousmodifications, including variations, additions, and omissions that maybe made in what has been described. Accordingly, it is intended thatthese modifications also be encompassed by the present invention andthat the scope of the present invention be limited solely by thebroadest interpretation that lawfully can be accorded the appendedclaims.

All patents, publications and references cited herein are hereby fullyincorporated by reference. In case of conflict between the presentdisclosure and incorporated patents, publications and references, thepresent disclosure should control.

1. A method of assessing responsiveness of cancer to treatment with aperipheral mu-opioid antagonist, comprising detecting the level ofexpression of the mu-opioid receptor (MOR) in a sample taken from asubject, and comparing the level of expression of the MOR in the samplewith a standard value, wherein an increased level of expression isindicative of treatment responsiveness to the peripheral mu-opioidantagonist.
 2. The method of claim 1, wherein the sample is obtainedfrom lung cancer tissue.
 3. The method of claim 1, wherein the increasedlevel of expression is indicative of the therapeutic efficacy of theantagonist.
 4. The method of claim 1, wherein the peripheral mu-opioidantagonist is a quaternary or non-quaternary derivative ofnoroxymorphone.
 5. A method of predicting a clinical response totreatment with a peripheral opioid antagonist of a subject in needthereof, comprising detecting the expression level of MOR in a samplefrom the subject, and comparing the expression level of MOR in thesample from the subject with the expression level from a subject not inneed of treatment, wherein increased expression of MOR is indicative ofresponsiveness to the opioid antagonist, and is predictive of a clinicalresponse to treatment of with an opioid antagonist.
 6. The method ofclaim 5, wherein the peripheral mu-opioid antagonist is a quaternary ornon-quaternary derivative of noroxymorphone.
 7. A method of assessinglung cancer prognosis, comprising detecting the MOR expression level ina specimen collected from a subject whose lung cancer prognosis is to beassessed; and comparing the level to a standard level, an elevated levelindicative that the subject is suffering from lung cancer or is at riskof recurrence of lung cancer and that the lung cancer is responsive toMOR antagonist treatment; and treating the subject with a mu-opioidantagonist.
 8. The method of claim 7, wherein the peripheral mu-opioidantagonist is a quaternary or non-quaternary derivative ofnoroxymorphone.
 9. A method of attenuating tumor growth of a cancer,metastasis of a cancer, or both, in which the cancer comprises cellsthat overexpress mu-opioid receptors, comprising administering to asubject in need thereof an effective amount of a peripheral mu-opioidantagonist to attenuate the growth and/or metastasis.
 10. A method ofachieving an effect in a subject, comprising administering to thesubject an effective amount of a peripheral mu-opioid antagonist,wherein the effect is inhibiting cellular hyperproliferation, treatingcancer, or inhibiting tumor growth.